CN117815265A - Gold palladium platinum nano enzyme bactericide and preparation and application thereof - Google Patents
Gold palladium platinum nano enzyme bactericide and preparation and application thereof Download PDFInfo
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- CN117815265A CN117815265A CN202311863965.9A CN202311863965A CN117815265A CN 117815265 A CN117815265 A CN 117815265A CN 202311863965 A CN202311863965 A CN 202311863965A CN 117815265 A CN117815265 A CN 117815265A
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- XEOCKQIQXJNTER-UHFFFAOYSA-N gold palladium platinum Chemical compound [Pd].[Pd].[Pd].[Pd].[Pd].[Pt].[Pt].[Pt].[Pt].[Pt].[Pt].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au].[Au] XEOCKQIQXJNTER-UHFFFAOYSA-N 0.000 title claims abstract description 106
- 230000000844 anti-bacterial effect Effects 0.000 title claims abstract description 105
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 102
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 102
- 239000003899 bactericide agent Substances 0.000 title claims abstract description 96
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- 230000001954 sterilising effect Effects 0.000 claims abstract description 21
- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 21
- 239000003814 drug Substances 0.000 claims abstract description 15
- 238000001727 in vivo Methods 0.000 claims abstract description 9
- 239000000243 solution Substances 0.000 claims description 143
- 239000010931 gold Substances 0.000 claims description 55
- 229910052737 gold Inorganic materials 0.000 claims description 55
- 239000003638 chemical reducing agent Substances 0.000 claims description 49
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 45
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 45
- 239000012266 salt solution Substances 0.000 claims description 42
- 238000000034 method Methods 0.000 claims description 31
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 26
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 20
- SDKPSXWGRWWLKR-UHFFFAOYSA-M sodium;9,10-dioxoanthracene-1-sulfonate Chemical compound [Na+].O=C1C2=CC=CC=C2C(=O)C2=C1C=CC=C2S(=O)(=O)[O-] SDKPSXWGRWWLKR-UHFFFAOYSA-M 0.000 claims description 20
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 claims description 18
- -1 gold ions Chemical class 0.000 claims description 18
- 230000032683 aging Effects 0.000 claims description 17
- WOWHHFRSBJGXCM-UHFFFAOYSA-M cetyltrimethylammonium chloride Chemical compound [Cl-].CCCCCCCCCCCCCCCC[N+](C)(C)C WOWHHFRSBJGXCM-UHFFFAOYSA-M 0.000 claims description 17
- 238000002156 mixing Methods 0.000 claims description 16
- 239000007974 sodium acetate buffer Substances 0.000 claims description 15
- 230000012010 growth Effects 0.000 claims description 14
- 150000002940 palladium Chemical class 0.000 claims description 13
- 150000003057 platinum Chemical class 0.000 claims description 13
- 208000035143 Bacterial infection Diseases 0.000 claims description 12
- 208000022362 bacterial infectious disease Diseases 0.000 claims description 12
- 238000005119 centrifugation Methods 0.000 claims description 11
- 230000001404 mediated effect Effects 0.000 claims description 11
- 239000011668 ascorbic acid Substances 0.000 claims description 10
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- 235000010323 ascorbic acid Nutrition 0.000 claims description 10
- 230000008569 process Effects 0.000 claims description 10
- 229910001112 rose gold Inorganic materials 0.000 claims description 10
- 239000002904 solvent Substances 0.000 claims description 10
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Substances [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 claims description 9
- 239000012279 sodium borohydride Substances 0.000 claims description 9
- 229910000033 sodium borohydride Inorganic materials 0.000 claims description 9
- 239000001509 sodium citrate Substances 0.000 claims description 8
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 8
- 238000005406 washing Methods 0.000 claims description 8
- 201000010099 disease Diseases 0.000 claims description 7
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 7
- KDLHZDBZIXYQEI-UHFFFAOYSA-N palladium Substances [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 claims description 7
- 229910052697 platinum Inorganic materials 0.000 claims description 7
- 230000002194 synthesizing effect Effects 0.000 claims description 7
- 239000012064 sodium phosphate buffer Substances 0.000 claims description 6
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- 229910052763 palladium Inorganic materials 0.000 claims description 5
- 230000000694 effects Effects 0.000 abstract description 29
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 abstract description 17
- 241000894006 Bacteria Species 0.000 abstract description 13
- 230000003197 catalytic effect Effects 0.000 abstract description 13
- 229910052751 metal Inorganic materials 0.000 abstract description 10
- 239000002184 metal Substances 0.000 abstract description 10
- 238000002474 experimental method Methods 0.000 abstract description 9
- 238000000338 in vitro Methods 0.000 abstract description 6
- 239000002086 nanomaterial Substances 0.000 abstract description 5
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- 239000000758 substrate Substances 0.000 abstract description 2
- 241000191967 Staphylococcus aureus Species 0.000 description 25
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 23
- RJQXTJLFIWVMTO-TYNCELHUSA-N Methicillin Chemical compound COC1=CC=CC(OC)=C1C(=O)N[C@@H]1C(=O)N2[C@@H](C(O)=O)C(C)(C)S[C@@H]21 RJQXTJLFIWVMTO-TYNCELHUSA-N 0.000 description 21
- 229960003085 meticillin Drugs 0.000 description 21
- 239000000047 product Substances 0.000 description 19
- 230000001580 bacterial effect Effects 0.000 description 16
- JRTYPQGPARWINR-UHFFFAOYSA-N palladium platinum Chemical compound [Pd].[Pt] JRTYPQGPARWINR-UHFFFAOYSA-N 0.000 description 12
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 11
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- 229960003165 vancomycin Drugs 0.000 description 8
- MYPYJXKWCTUITO-UHFFFAOYSA-N vancomycin Natural products O1C(C(=C2)Cl)=CC=C2C(O)C(C(NC(C2=CC(O)=CC(O)=C2C=2C(O)=CC=C3C=2)C(O)=O)=O)NC(=O)C3NC(=O)C2NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(CC(C)C)NC)C(O)C(C=C3Cl)=CC=C3OC3=CC2=CC1=C3OC1OC(CO)C(O)C(O)C1OC1CC(C)(N)C(O)C(C)O1 MYPYJXKWCTUITO-UHFFFAOYSA-N 0.000 description 8
- MYPYJXKWCTUITO-LYRMYLQWSA-O vancomycin(1+) Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C2C=C3C=C1OC1=CC=C(C=C1Cl)[C@@H](O)[C@H](C(N[C@@H](CC(N)=O)C(=O)N[C@H]3C(=O)N[C@H]1C(=O)N[C@H](C(N[C@@H](C3=CC(O)=CC(O)=C3C=3C(O)=CC=C1C=3)C([O-])=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)O2)=O)NC(=O)[C@@H](CC(C)C)[NH2+]C)[C@H]1C[C@](C)([NH3+])[C@H](O)[C@H](C)O1 MYPYJXKWCTUITO-LYRMYLQWSA-O 0.000 description 8
- 206010018910 Haemolysis Diseases 0.000 description 7
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 7
- 241000699670 Mus sp. Species 0.000 description 7
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- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 6
- 229910052700 potassium Inorganic materials 0.000 description 6
- 239000011591 potassium Substances 0.000 description 6
- VCUVETGKTILCLC-UHFFFAOYSA-N 5,5-dimethyl-1-pyrroline N-oxide Chemical compound CC1(C)CCC=[N+]1[O-] VCUVETGKTILCLC-UHFFFAOYSA-N 0.000 description 5
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- 241000588626 Acinetobacter baumannii Species 0.000 description 4
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 4
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 4
- 241000194032 Enterococcus faecalis Species 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 4
- 241000588747 Klebsiella pneumoniae Species 0.000 description 4
- 241000589517 Pseudomonas aeruginosa Species 0.000 description 4
- 206010052428 Wound Diseases 0.000 description 4
- 208000027418 Wounds and injury Diseases 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- 229940032049 enterococcus faecalis Drugs 0.000 description 4
- TUJKJAMUKRIRHC-UHFFFAOYSA-N hydroxyl Chemical compound [OH] TUJKJAMUKRIRHC-UHFFFAOYSA-N 0.000 description 4
- 208000015181 infectious disease Diseases 0.000 description 4
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 3
- 239000006137 Luria-Bertani broth Substances 0.000 description 3
- 102000003992 Peroxidases Human genes 0.000 description 3
- 239000002202 Polyethylene glycol Substances 0.000 description 3
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 239000003242 anti bacterial agent Substances 0.000 description 3
- 239000007864 aqueous solution Substances 0.000 description 3
- 244000052616 bacterial pathogen Species 0.000 description 3
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- 229940124597 therapeutic agent Drugs 0.000 description 3
- 229910003803 Gold(III) chloride Inorganic materials 0.000 description 2
- 108700020962 Peroxidase Proteins 0.000 description 2
- 206010048038 Wound infection Diseases 0.000 description 2
- 238000000862 absorption spectrum Methods 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- WPEJSSRSFRWYJB-UHFFFAOYSA-K azanium;tetrachlorogold(1-) Chemical compound [NH4+].[Cl-].[Cl-].[Cl-].[Cl-].[Au+3] WPEJSSRSFRWYJB-UHFFFAOYSA-K 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
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- 238000012512 characterization method Methods 0.000 description 2
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- 238000010586 diagram Methods 0.000 description 2
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- 239000000417 fungicide Substances 0.000 description 2
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- 150000002343 gold Chemical class 0.000 description 2
- RJHLTVSLYWWTEF-UHFFFAOYSA-K gold trichloride Chemical compound Cl[Au](Cl)Cl RJHLTVSLYWWTEF-UHFFFAOYSA-K 0.000 description 2
- 229940076131 gold trichloride Drugs 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
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- 239000000203 mixture Substances 0.000 description 2
- MUJIDPITZJWBSW-UHFFFAOYSA-N palladium(2+) Chemical compound [Pd+2] MUJIDPITZJWBSW-UHFFFAOYSA-N 0.000 description 2
- PIBWKRNGBLPSSY-UHFFFAOYSA-L palladium(II) chloride Chemical compound Cl[Pd]Cl PIBWKRNGBLPSSY-UHFFFAOYSA-L 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 239000003642 reactive oxygen metabolite Substances 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- FBEIPJNQGITEBL-UHFFFAOYSA-J tetrachloroplatinum Chemical compound Cl[Pt](Cl)(Cl)Cl FBEIPJNQGITEBL-UHFFFAOYSA-J 0.000 description 2
- RKMGAJGJIURJSJ-UHFFFAOYSA-N 2,2,6,6-Tetramethylpiperidine Substances CC1(C)CCCC(C)(C)N1 RKMGAJGJIURJSJ-UHFFFAOYSA-N 0.000 description 1
- KZMAWJRXKGLWGS-UHFFFAOYSA-N 2-chloro-n-[4-(4-methoxyphenyl)-1,3-thiazol-2-yl]-n-(3-methoxypropyl)acetamide Chemical compound S1C(N(C(=O)CCl)CCCOC)=NC(C=2C=CC(OC)=CC=2)=C1 KZMAWJRXKGLWGS-UHFFFAOYSA-N 0.000 description 1
- 238000009631 Broth culture Methods 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
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Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention belongs to the technical field of medicine and nano materials, and particularly discloses a gold-palladium-platinum nano enzyme bactericide and preparation and application thereof. The bactericide comprises gold-palladium-platinum nano-enzyme, and the concentration of the gold-palladium-platinum nano-enzyme in the bactericide is less than or equal to 20.83 mug/mL; the bactericide has broad-spectrum bactericidal performance. Proved by repeated experiments, the gold-palladium-platinum nano-enzyme in the bactericide provided by the invention has high-efficiency double-enzyme catalytic activity of peroxidase-like enzyme and oxidase-like enzyme, and generates various free radicals through double-enzyme cascade catalytic substrates so as to realize high-efficiency sterilization. The gold-palladium-platinum nano enzyme bactericide not only solves the problems of limited catalytic activity and single enzyme activity of single metal nano enzyme, but also has good biocompatibility, and is suitable for in-vitro and in-vivo drug-resistant bacteria treatment. In addition, the high-efficiency sterilization effect can be achieved without adding hydrogen peroxide during treatment, the skin is not damaged, and the problems of in-vivo sterilization effect and patient compliance are greatly improved.
Description
Technical Field
The invention relates to the technical field of medical science and nano materials, in particular to the technical field of medical sterilization nano materials, and particularly relates to a gold-palladium-platinum nano enzyme bactericide, and preparation and application thereof.
Background
Infections caused by resistant bacteria are a major health problem worldwide, with super resistant bacterial infections, represented by methicillin-resistant staphylococcus aureus (Methicillin Resistant Staphylococcus Aureus, MRSA), being the clinically common more infectious bacterial infections. Antibiotics are a common method in clinic for treating bacterial infection, but the long-term use of antibiotics leads to the enhancement of bacterial resistance and the appearance of super-resistant bacteria, which seriously threatens human health. Therefore, there is an urgent need to find a new fungicide to combat drug-resistant bacterial infections.
Nano-enzymes can play a role in sterilization by catalyzing the generation of reactive oxygen species (Reactive Oxygen Species, ROS), and studies on their use as novel bactericides have been reported. Among them, a representative single metal nano-enzyme, such as gold nano-enzyme, has activity similar to peroxide nano-enzyme, and can catalyze hydrogen peroxide to generate hydroxyl radical to sterilize. However, the bactericidal efficacy of single metal nanoenzymes is still limited by insufficient catalytic activity. In addition, when the peroxidase-like nano enzyme is used for sterilization treatment, hydrogen peroxide needs to be added, and the problems of skin compliance and in-vivo sterilization limitation exist. Meanwhile, the catalytic activity of the nano-enzyme serving as an artificial nano-material is influenced by various factors. Wherein, the composition structure of the nano material plays a key role in the catalytic activity of the nano enzyme. Compared with single-atom metal nano enzyme, the multi-metal nano enzyme has multi-metal synergistic enhancement of electron transfer in nano enzyme, and simultaneously exerts different enzyme activities of multiple metals, namely, the multi-enzyme activity is integrated, and all enzyme activities can skillfully form a cascade reaction system, so that self-sufficiency of intermediate products is realized, a closed loop of the reaction is formed, and the nano enzyme has stronger catalytic capability and higher sterilization efficiency. Therefore, based on the specific properties and advantages of the multi-metal nano enzyme, a more efficient bactericide can be developed.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, the present invention aims to provide a gold-palladium-platinum nano enzyme bactericide, and preparation and application thereof, which are used for solving the problems of poor bactericidal efficacy, skin compliance and in vivo sterilization limitation caused by limited catalytic activity due to a single-atom metal nano enzyme structure in the prior art.
In order to achieve the above object, a first aspect of the present invention provides a gold-palladium-platinum nano-enzyme bactericide, which comprises gold-palladium-platinum nano-enzyme, wherein the concentration of gold-palladium-platinum nano-enzyme in the bactericide is less than or equal to 20.83 μg/mL; the bactericide has broad-spectrum bactericidal performance.
Compared with the prior art, the invention has the following beneficial effects:
1. the gold-palladium-platinum nano enzyme bactericide provided by the invention has double-enzyme catalytic activity of peroxidase-like and oxidase-like, and can catalyze substrates to generate various free radicals for sterilization through double-enzyme cascade reaction, and is specifically characterized in that the capability of the gold-palladium-platinum nano enzyme for catalyzing oxygen to generate hydrogen peroxide and the capability of the peroxidase-like enzyme for catalyzing hydrogen peroxide to generate hydroxyl free radicals are combined to form a cascade enzyme catalytic system, so that the catalytic performance of the nano enzyme is improved, and the efficient sterilization effect is realized. The nano-enzyme has high-efficiency enzyme activity, integrates multi-enzyme activity, can skillfully form a cascade reaction system among the enzyme activities, realizes self-sufficiency of intermediate products, forms a closed loop of reaction, and greatly improves the problem of limited catalytic activity of the single-metal nano-enzyme.
2. The gold-palladium-platinum nano enzyme bactericide provided by the invention not only can be used for high-efficiency sterilization, but also has good biocompatibility, is suitable for in-vivo and in-vitro sterilization treatment, and improves the skin compliance of patients.
3. The gold-palladium-platinum nano enzyme bactericide provided by the invention is prepared by an etching-regenerating synthetic strategy, and the gold-palladium-platinum nano enzyme bactericide with a multi-layer structure can be prepared by simply changing the dosage of gold seed solution, so that the preparation method is simple, the cost is low, and a very promising choice is provided for avoiding drug resistance generation and clinically resisting bacteria treatment.
Further, the concentration of the gold-palladium-platinum nano-enzyme is 7.0-31.25. Mu.g/mL, preferably 7.81-20.83. Mu.g/mL.
Further, the gold palladium platinum nano-enzyme has a spherical outline, and the average size is 40-110 nm, preferably 40-50 nm.
Further, the bactericide further comprises a solvent selected from at least one of water, acetic acid/sodium acetate buffer and phosphate buffer.
Further, the preparation method of the gold-palladium-platinum nano enzyme comprises the following steps:
a1, synthesizing gold seeds based on a seed-mediated growth method, and preparing a wine-red gold seed solution;
a2, mixing the gold seed solution with a hexadecyl trimethyl ammonium chloride solution, a palladium salt solution, a platinum salt solution and a reducing agent solution, and standing and aging at 50-70 ℃; and after the ageing is finished, centrifugally collecting a product, and washing the product to obtain the gold-palladium-platinum nano enzyme.
Further, the step A1 synthesizes gold seeds based on a seed-mediated growth method, and the process of preparing the gold seed solution with the color of reddish wine comprises the following steps:
a11, mixing a gold salt solution and a cetyltrimethylammonium bromide solution, adding an ice-cold reducing agent solution, and immediately turning the solution into brown to obtain an initial small gold seed solution;
a12, mixing the gold salt solution, the cetyltrimethylammonium chloride solution and the reducing agent solution, adding the initial small gold seed solution, standing, centrifugally collecting the product, and dispersing the product in water to obtain the wine-red gold seed solution.
Further, in the steps a11 and a12, the reducing agent is at least one selected from sodium borohydride, ascorbic acid and sodium citrate.
Further, in the step A11, the molar ratio of the gold ions in the gold salt solution, the cetyltrimethylammonium bromide solution and the reducing agent solution is 2-3:800-1200:4-8.
Further, in the step A11, the ice-cold reducing agent solution is a reducing agent solution at 4℃or through an ice-water bath.
Further, in the step A12, the molar ratio of the gold ions in the gold salt solution, the cetyltrimethylammonium bromide solution and the reducing agent solution is 1-3:600-1000:200-400.
Further, in the step A12, the standing time is 20 to 40 minutes.
Further, in the step A12, the centrifugation time is 60 to 80 minutes, and the rotation speed is 14000 to 20000rpm.
Further, in the step A2, the molar ratio of the gold seed solution, the cetyltrimethylammonium chloride solution, the palladium ions in the palladium salt solution and the platinum ions in the platinum salt solution to the reducing agent solution is 0.05-1.2:30-50:1:1:80-120.
Further, in the step A2, the aging time is 2-4 hours.
In the step A2, the centrifugation time is 8-15 min, and the rotation speed is 14000-20000 rpm.
Further, in the step A2, the reducing agent is at least one selected from sodium borohydride, ascorbic acid and sodium citrate.
The second aspect of the invention provides a preparation method of the golden palladium platinum nano enzyme bactericide according to the first aspect, which comprises the following steps:
b1, synthesizing gold seeds based on a seed-mediated growth method, and preparing a wine-red gold seed solution;
b2, mixing the gold seed solution with a hexadecyl trimethyl ammonium chloride solution, a palladium salt solution, a platinum salt solution and a reducing agent solution, and standing and aging at 50-70 ℃; centrifuging and collecting a product after aging is finished, and washing the product to obtain the gold-palladium-platinum nano enzyme;
and B3, adding the gold-palladium-platinum nano enzyme into a solvent to prepare the bactericide.
Further, the step B1 is based on a seed-mediated growth method for synthesizing gold seeds, and the process for preparing the golden seed solution with the color of reddish wine comprises the following steps:
b11, mixing a gold salt solution and a cetyltrimethylammonium bromide solution, adding an ice-cold reducing agent solution, and immediately turning the solution into brown to obtain an initial small gold seed solution;
and B12, mixing a gold salt solution, a cetyltrimethylammonium chloride solution and a reducing agent solution, adding an initial small gold seed solution, standing, centrifugally collecting a product, and dispersing the product in water to obtain a wine-red gold seed solution.
Further, in the steps B11 and B12, the reducing agent is at least one selected from sodium borohydride, ascorbic acid and sodium citrate.
Further, in the step B11, the molar ratio of the gold ions in the gold salt solution, the cetyltrimethylammonium bromide solution and the reducing agent solution is 2-3:800-1200:4-8.
Further, in the step B11, the ice-cold reducing agent solution is a reducing agent solution at 4 ℃ or through an ice-water bath.
Further, in the step B12, the molar ratio of the gold ions in the gold salt solution, the cetyltrimethylammonium bromide solution and the reducing agent solution is 1-3:600-1000:200-400.
Further, in the step B12, the standing time is 20 to 40 minutes.
In step B12, the centrifugation time is 60 to 80 minutes, and the rotation speed is 14000 to 20000rpm.
Further, in the step B2, the molar ratio of the gold seed solution, the cetyltrimethylammonium chloride solution, the palladium ions in the palladium salt solution and the platinum ions in the platinum salt solution to the reducing agent solution is 0.05-1.2:30-50:1:1:80-120.
Further, in the step B2, the aging time is 2-4 hours.
In step B2, the centrifugation time is 8-15 min, and the rotation speed is 14000-20000 rpm.
Further, in the step B2, the reducing agent is at least one selected from sodium borohydride, ascorbic acid and sodium citrate.
Further, in the step B3, the solvent is at least one selected from water, acetic acid/sodium acetate buffer and phosphate buffer.
According to a third aspect of the invention, there is provided the use of a nano-enzyme bactericide of gold, palladium and platinum according to the first aspect and/or a nano-enzyme bactericide of gold, palladium and platinum prepared according to the method of the second aspect as or in the preparation of a medicament and/or bactericide for the treatment of a disease caused by bacterial infection.
Further, the medicine and/or bactericide for treating diseases caused by bacterial infection is applied to in-vitro sterilization.
Further, the bacteria that the bactericide can kill include gram-positive bacteria and gram-negative bacteria.
Drawings
FIG. 1 shows the oxidase-like activity characterization of the gold-palladium-platinum nano-enzyme bactericide of example 1 of the present invention. Wherein (a) is a solution of 3,3', 5' -tetramethylbenzidine; (b) (c) and (d) are ultraviolet-visible spectrograms of the gold palladium platinum nano enzyme bactericides with the sizes of 106.54nm, 79.76nm and 46.43nm after the 3,3', 5' -tetramethyl benzidine solution is added.
FIG. 2 shows the characterization of peroxidase-like activity of the gold-palladium-platinum nano-enzyme bactericide of example 1 of the present invention. Wherein, (a) is a hydrogen peroxide solution and a 3,3', 5' -tetramethylbenzidine solution; (b) (c) and (d) are ultraviolet-visible spectrograms of the gold palladium platinum nano enzyme bactericides with the sizes of 106.54nm, 79.76nm and 46.43nm after adding 3,3', 5' -tetramethyl benzidine solution and hydrogen peroxide solution.
FIG. 3 shows a scanning electron microscope image of a gold-palladium-platinum nano-enzyme bactericide of a size of 46.43nm in example 1 of the present invention. Wherein, (a) is a low-power imaging diagram of the golden palladium platinum nano enzyme bactericide; (b) is a high power imaging diagram of the golden palladium platinum nano enzyme bactericide.
FIG. 4 shows the presentElectron spin resonance spectrum of active oxygen generated by gold-palladium-platinum nano enzyme bactericide in invention example 2. Wherein (a), (b) and (c) are each superoxide anion radical (O) 2 · (-) singlet oxygen molecules 1 O 2 ) Electron spin resonance spectra for hydroxyl radical signal (·oh) detection.
Fig. 5 shows the effect of the gold-palladium-platinum nano-enzyme bactericide in example 3 of the present invention on killing methicillin-resistant staphylococcus aureus in vitro. Wherein, the growth conditions of agar plate colonies after the methicillin-resistant staphylococcus aureus is incubated with 0.9 percent NaCl, acetic acid/sodium acetate buffer solution, 4 percent polyethylene glycol, vancomycin and gold palladium platinum nano enzyme bactericides with different concentrations are respectively from left to right.
FIG. 6 shows a transmission electron micrograph of a gold-palladium-platinum nanoenzyme breaking down methicillin-resistant Staphylococcus aureus cell wall and cell membrane in example 3 of the present invention. Wherein, (a) is a transmission electron microscope image of methicillin-resistant staphylococcus aureus incubated with 0.9% NaCl; (b) Is a transmission electron microscope image of methicillin-resistant staphylococcus aureus and gold palladium platinum nano enzyme after co-incubation.
FIG. 7 shows the effect of the gold-palladium-platinum nano-enzyme bactericide in example 3 of the present invention on killing enterococcus faecalis, staphylococcus aureus, escherichia coli, klebsiella pneumoniae, acinetobacter baumannii and Pseudomonas aeruginosa.
FIG. 8 shows cytotoxicity evaluation of the gold-palladium-platinum nano-enzyme bactericide in example 4 of the present invention. Wherein, the bar graph and the inset are negative control group (0.9% NaCl), golden palladium platinum nano enzyme bactericide with different concentrations (8.93, 10.42, 12.50, 15.63, 20.83 and 31.25 mug/mL) and positive control group (H) in turn from left to right 2 O) the rate of hemolysis after mixing with blood and the color change of the sample.
Fig. 9 shows photographs of wound wounds of mice treated with phosphate buffer, vancomycin and gold palladium platinum nano enzyme bactericide, respectively, in example 5 of the present invention at corresponding time points.
Detailed Description
Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention.
The invention provides a gold-palladium-platinum nano enzyme bactericide, which comprises gold-palladium-platinum nano enzyme, wherein the concentration of the gold-palladium-platinum nano enzyme in the bactericide is less than or equal to 20.83 mug/mL; the bactericide has broad-spectrum bactericidal performance.
In some embodiments, the concentration of the gold palladium platinum nano-enzyme bactericide is 7.0 to 20.83 μg/mL, preferably 7.81 to 20.83 μg/mL, such as 7.81, 8.0, 8.93, 9.0, 10.0, 10.42, 11.0, 12.0, 12.50, 13.0, 14.0, 15.0, 15.63, 16.0, 17.0, 18.0, 19.0, 20.0, 20.50, 20.83 μg/mL, etc.
In some embodiments, the gold palladium platinum nanoenzyme germicides have a spherical profile with an average size of 40-110 nm, preferably 40-50 nm and/or 100-110 nm, such as 40, 42, 45, 46.43, 48, 50, 70, 79.63, 80, 90, 100, 102, 105, 108, 110nm, etc.
In some embodiments, the germicide further comprises a solvent selected from at least one of water, acetic acid/sodium acetate buffer, and phosphate buffer; wherein the water is selected from deionized water, ultrapure water, etc.; acetic acid/sodium acetate buffer with ph=4.5, phosphate buffer with pH 7.0-7.8 and 0.01 m.
In some embodiments, the preparation method of the gold-palladium-platinum nano enzyme bactericide comprises the following steps:
a1, synthesizing gold seeds based on a seed-mediated growth method, and preparing a wine-red gold seed solution;
a2, mixing the gold seed solution with a hexadecyl trimethyl ammonium chloride solution, a palladium salt solution, a platinum salt solution and a reducing agent solution, and standing and aging at 50-70 ℃; and after the ageing is finished, centrifugally collecting a product, and washing the product to obtain the gold-palladium-platinum nano enzyme.
In some embodiments, the step A1 synthesizes gold seeds based on a seed-mediated growth method, and the process of formulating into a reddish gold seed solution comprises the steps of:
a11, mixing a gold salt solution and a cetyltrimethylammonium bromide solution, adding an ice-cold reducing agent solution, and immediately turning the solution into brown to obtain an initial small gold seed solution;
a12, mixing the gold salt solution, the cetyltrimethylammonium chloride solution and the reducing agent solution, adding the initial small gold seed solution, standing, centrifugally collecting the product, and dispersing the product in water to obtain the wine-red gold seed solution.
In some embodiments, in steps a11, a12 and A2, the gold salts include, but are not limited to, chloroauric acid, gold trichloride, sodium chloroaurate, potassium chloroaurate, ammonium chloroaurate, etc., the palladium salts include, but are not limited to, palladium chloride acid, sodium chloropalladate, potassium chloropalladate, ammonium chloropalladate, etc., and the platinum salts include, but are not limited to, platinum tetrachloride, chloroplatinic acid, sodium chloroplatinate, potassium chloroplatinate, ammonium chloroplatinate, etc., and the reducing agents include, but are not limited to, sodium borohydride, ascorbic acid, sodium citrate, etc.
In some embodiments, in the step a11, the molar ratio of the gold ions in the gold salt solution, the cetyltrimethylammonium bromide solution and the reducing agent solution is 2-3:800-1200:4-8, such as 2:800:4, 2:1000:4, 2:1200:4, 2.5:800:6, 2.5:1000:6, 2.5:1200:6, 3:800:8, 3:1000:8, 3:1200:8, etc.
In some embodiments, in step a11, the ice-cold reducing agent solution is a reducing agent solution at 4 ℃ or through an ice-water bath.
In some embodiments, in the step a12, the molar ratio of the gold ions in the gold salt solution, the cetyltrimethylammonium bromide solution and the reducing agent solution is 1-3:600-1000:200-400, such as 1:600:200, 1:800:200, 1:1000:200, 2:600:200, 2:800:300, 1:1000:200, 3:600:200, 3:800:200, 3:1000:200, etc.
In some embodiments, in step a12, the resting time is 20-40 min, e.g., 20, 25, 30, 35, 40min, etc.
In some embodiments, in step a12, the centrifugation time is 60 to 80min, such as 60, 65, 70, 75, 80min, etc.; the rotation speed is 14000 to 20000rpm, for example, 14000, 15000, 16000, 17000, 18000, 19000, 20000rpm, or the like.
In some embodiments, in the step A2, the molar ratio of the gold seed, the cetyltrimethylammonium chloride solution, the palladium ion in the palladium salt solution, the platinum ion in the platinum salt solution, and the reducing agent solution is 0.05-1.2:30-50:1:1:80-120, for example, 0.05:30:1:1:80, 0.05:40:1:1:80, 0.05:50:1:1:80, 0.1:30:1:1:80, 0.1:40:1:1:80, 0.1:50:1:1:80, 1:40:1:1:100, 1.2:30:1:1:120, 1.2:40:1:1:120, 1.2:50:1:1:120, and the like.
In some embodiments, in step A2, the aging temperature is 50 to 70 ℃, e.g., 50, 55, 60, 65, 70 ℃, etc.
In some embodiments, in step A2, the aging time is 2 to 4 hours, such as 2, 2.5, 3, 3.5, 4 hours, etc.
In some embodiments, in step A2, the centrifugation time is 8 to 15min, e.g., 8, 9, 10, 12, 15min, etc.; the rotation speed is 14000 to 20000rpm, for example, 14000, 15000, 16000, 17000, 18000, 19000, 20000rpm, or the like.
Another embodiment of the present invention provides a method for preparing a gold-palladium-platinum nano enzyme bactericide according to the above embodiment/example, comprising the following steps:
b1, synthesizing gold seeds based on a seed-mediated growth method, and preparing a wine-red gold seed solution;
b2, mixing the gold seed solution with a hexadecyl trimethyl ammonium chloride solution, a palladium salt solution, a platinum salt solution and a reducing agent solution, and standing and aging at 50-70 ℃; centrifuging and collecting a product after aging is finished, and washing the product to obtain the gold-palladium-platinum nano enzyme;
and B3, adding the gold-palladium-platinum nano enzyme into a solvent to prepare the bactericide.
In some embodiments, the step B1 synthesizes gold seeds based on a seed-mediated growth method, and the process of formulating into a reddish gold seed solution comprises the steps of:
b11, mixing a gold salt solution and a cetyltrimethylammonium bromide solution, adding an ice-cold reducing agent solution, and immediately turning the solution into brown to obtain an initial small gold seed solution;
and B12, mixing a gold salt solution, a cetyltrimethylammonium chloride solution and a reducing agent solution, adding an initial small gold seed solution, standing, centrifugally collecting a product, and dispersing the product in water to obtain a wine-red gold seed solution.
In some embodiments, in steps B11, B12 and B2, the gold salts include, but are not limited to, chloroauric acid, gold trichloride, sodium chloroaurate, potassium chloroaurate, ammonium chloroaurate, etc., the palladium salts include, but are not limited to, palladium chloride acid, sodium chloropalladate, potassium chloropalladate, ammonium chloropalladate, etc., and the platinum salts include, but are not limited to, platinum tetrachloride, chloroplatinic acid, sodium chloroplatinate, potassium chloroplatinate, ammonium chloroplatinate, etc., and the reducing agents include, but are not limited to, sodium borohydride, ascorbic acid, sodium citrate, etc.
In some embodiments, in the step B11, the molar ratio of the gold ions in the gold salt solution, the cetyltrimethylammonium bromide solution and the reducing agent solution is 2-3:800-1200:4-8, such as 2:800:4, 2:1000:4, 2:1200:4, 2.5:800:6, 2.5:1000:6, 2.5:1200:6, 3:800:8, 3:1000:8, 3:1200:8, etc.
In some embodiments, in step B11, the ice-cold reducing agent solution is a reducing agent solution at 4 ℃ or through an ice-water bath.
In some embodiments, in the step B12, the molar ratio of the gold ions in the gold salt solution, the cetyltrimethylammonium bromide solution and the reducing agent solution is 1-3:600-1000:200-400, such as 1:600:200, 1:800:200, 1:1000:200, 2:600:200, 2:800:300, 1:1000:200, 3:600:200, 3:800:200, 3:1000:200, etc.
In some embodiments, the standing time in step B12 is 20 to 40min, such as 20, 25, 30, 35, 40min, etc.
In some embodiments, in step B12, the centrifugation time is 60 to 80 minutes, such as 60, 65, 70, 75, 80 minutes, etc.; the rotation speed is 14000 to 20000rpm, for example, 14000, 15000, 16000, 17000, 18000, 19000, 20000rpm, or the like.
In some embodiments, in the step B2, the molar ratio of the gold seed, the cetyltrimethylammonium chloride solution, the palladium ion in the palladium salt solution, the platinum ion in the platinum salt solution, and the reducing agent solution is 0.05-1.2:30-50:1:1:80-120, for example, 0.05:30:1:1:80, 0.05:40:1:1:80, 0.05:50:1:1:80, 0.1:30:1:1:80, 0.1:40:1:1:80, 0.1:50:1:1:80, 1:40:1:1:100, 1.2:30:1:1:120, 1.2:40:1:1:120, 1.2:50:1:1:120, and the like.
In some embodiments, in step B2, the aging temperature is 50 to 70 ℃, e.g., 50, 55, 60, 65, 70 ℃, etc.
In some embodiments, in step B2, the aging time is 2 to 4 hours, such as 2, 2.5, 3, 3.5, 4 hours, etc.
In some embodiments, in step B2, the centrifugation time is 8 to 15min, e.g., 8, 9, 10, 12, 15min, etc.; the rotation speed is 14000 to 20000rpm, for example, 14000, 15000, 16000, 17000, 18000, 19000, 20000rpm, or the like.
In some embodiments, in step B3, the solvent is selected from at least one of water, acetic acid/sodium acetate buffer, and phosphate buffer.
It should be noted that the initial small gold seed solution, cetyltrimethylammonium chloride solution, cetyltrimethylammonium bromide solution, palladium salt solution, platinum salt solution, and reducing agent solution in the above embodiment/example of the present invention are prepared by using water or other solvents, but are not limited thereto.
In another embodiment of the present invention, the gold-palladium-platinum nano-enzyme bactericide according to the above embodiment/example can be used as a medicament and/or bactericide for treating a disease caused by bacterial infection or used for preparing a medicament and/or bactericide for treating a disease caused by bacterial infection.
In some embodiments, the medicament and/or fungicide for treating diseases caused by bacterial infection is applied to in-vitro sterilization and has good biocompatibility. In addition, the golden palladium platinum nano enzyme bactericide can play a role in high-efficiency sterilization without adding hydrogen peroxide when treating drug-resistant bacterial infection, does not hurt skin, and greatly improves the problems of in-vivo sterilization curative effect and patient compliance.
In some embodiments, the bacteria that the germicide is capable of killing include, but are not limited to, gram positive and gram negative bacteria, such as methicillin-resistant staphylococcus aureus, enterococcus faecalis, staphylococcus aureus, escherichia coli, klebsiella pneumoniae, acinetobacter baumannii, pseudomonas aeruginosa.
The following specific exemplary examples illustrate the invention in detail. It is also to be understood that the following examples are given solely for the purpose of illustration and are not to be construed as limitations upon the scope of the invention, as many insubstantial modifications and variations are within the scope of the invention as would be apparent to those skilled in the art in light of the foregoing disclosure. The specific process parameters and the like described below are also merely examples of suitable ranges, i.e., one skilled in the art can make a suitable selection from the description herein and are not intended to be limited to the specific values described below.
Example 1
The gold-palladium-platinum nano enzyme bactericide is prepared in the embodiment, and the activity of peroxidases and peroxidases is researched, and the specific implementation process is as follows:
1. experimental method
(1) Preparation of gold palladium platinum nano enzyme bactericide
Step one: synthesis of gold seeds
The method for synthesizing gold seeds based on seed-mediated growth comprises the following steps: 5mL of 0.5mM chloroauric acid solution, 5mL of 200mM cetyltrimethylammonium bromide solution, and ice-cooled 0.6mL of 10mM sodium borohydride solution were added, and the solution became brown immediately, thus obtaining the initial small gold seeds. 4mL of 0.5mM chloroauric acid solution, 4mL of 200mM cetyltrimethylammonium chloride solution, 3mL of 100mM ascorbic acid solution were mixed in a glass vial (20 mL), 50. Mu.L of the initial small gold seed was added, left standing for 30min, and then centrifuged at 15000rpm for 70min, and the product was dispersed in 2mL of water to prepare a reddish-colored gold seed solution.
Step two: synthesis of gold palladium platinum nano enzyme bactericide
mu.L of the gold seed solution prepared in step one, 2mL of 20mM cetyltrimethylammonium bromide solution, 50. Mu.L of 20mM sodium chloropalladate solution, 50. Mu.L of 20mM chloroplatinic acid solution, 1mL of 100mM ascorbic acid solution were taken, mixed in a glass vial (10 mL), and then aged in a water bath at 60℃for 3 hours. Centrifuging at 16000rpm for 10min, collecting the product, washing with water once, and dispersing the product in 1mL of water to obtain the gold-palladium-platinum nano enzyme. Increasing the volume of the gold seed solution from 20 mu L to 100 mu L and 500 mu L respectively to obtain three gold-palladium-platinum nano enzymes with different sizes, wherein the sizes of the gold-palladium-platinum nano enzymes are as follows: 106.54nm, 79.76nm and 46.43nm.
(2) Detection of oxidase-like activity and peroxidase-like activity of gold-palladium-platinum nano enzyme bactericide
Step one, measuring the activity of the oxidase: respectively taking 5 mu L of three gold-palladium-platinum nano enzyme solutions with different sizes, 400 mu L of acetic acid/sodium acetate buffer solution, 400 mu L of 0.1mM 3,3', 5' -tetramethyl benzidine solution, adding water to dilute the solution to a total volume of 2mL, taking water as a blank control instead of a sample, and measuring ultraviolet-visible absorption after reacting for 15 minutes.
Step two, measuring the activity of the similar peroxidase: respectively taking 5 mu L of three gold-palladium-platinum nano enzyme solutions with different sizes, 400 mu L of pH=4 acetic acid/sodium acetate buffer solution, 400 mu L of 10mM hydrogen peroxide solution, 400 mu L of 0.1mM 3,3', 5' -tetramethyl benzidine solution, adding water to dilute the solution to a total volume of 2mL, taking water as a blank control to replace a sample, reacting for 15 minutes, and measuring the ultraviolet-visible absorption at 500-800 nm by an ultraviolet-visible spectrophotometer.
2. Analysis of experimental results
As shown in FIG. 1, the oxidase-like activity of the gold palladium platinum nano enzyme bactericide with the size of 46.43nm can be found to be optimal by comparing ultraviolet-visible absorption spectra.
As shown in FIG. 2, the peroxidase-like activity of the gold palladium platinum nano enzyme bactericide with the size of 46.43nm can be found to be optimal by comparing ultraviolet-visible absorption spectra.
As shown in fig. 3, as can be seen from fig. 3 (a), the small-size gold-palladium-platinum nano enzyme bactericide particles are in a spherical outline, are uniformly dispersed under a low-power mirror, and as can be seen from fig. 3 (b), a single sphere is in a loose structure consisting of a plurality of small grains, so that the gold-palladium-platinum nano enzyme bactericide particles have great catalytic potential.
Example 2
The sterilization performance of the gold palladium platinum nano enzyme bactericide with the size of 46.43nm prepared in the example 1 is evaluated in the embodiment, and the specific implementation process is as follows:
in the following experimental method, DMPO used refers to 5, 5-dimethyl-1-pyrroline-N-oxide; the TEMP used refers to 4-amino-2, 6-tetramethylpiperidine. DMPO as hydroxyl radical (. OH) and superoxide anion radical (O) 2 ·- ) Specific capturing agent, TEMP is used as singlet oxygen molecule 1 O 2 ) And (3) a specific capture agent, wherein an electron paramagnetic resonance instrument is used for testing the characteristic absorption of a corresponding addition object and testing a free radical signal.
1. Experimental method
Test one: under the condition of introducing oxygen, an electron paramagnetic resonance instrument is used for testing the superoxide anion free radical signal in the mixed aqueous solution of the system free radical capturing agent DMPO and the gold palladium platinum nano enzyme bactericide.
And II, testing: under the condition of introducing oxygen, an electron paramagnetic resonance instrument is used for testing a singlet oxygen molecular signal in a mixed aqueous solution of a system free radical capturing agent TEMP and a gold palladium platinum nano enzyme bactericide.
And (3) testing: under the condition of introducing oxygen, an electron paramagnetic resonance instrument is used for testing a hydroxyl radical signal in a mixed aqueous solution of a system radical trapping agent DMPO and a gold palladium platinum nano enzyme bactericide.
2. Analysis of experimental results
As shown in fig. 4, the test shows that under the condition of oxygen introduction, the electron paramagnetic resonance instrument detects that the gold palladium platinum nano enzyme bactericide can rapidly generate various active oxygen, including superoxide anion free radicals, singlet oxygen molecules and hydroxyl free radical signals within 10 minutes. The results show that the gold-palladium-platinum nano enzyme bactericide has high-efficiency capability of catalyzing the generation of active oxygen and has excellent bactericidal effect.
Example 3
The gold palladium platinum nano enzyme bactericide with the size of 46.43nm prepared in the embodiment 1 is used for in vitro killing of drug-resistant bacteria, and the specific implementation process is as follows:
1. experimental method
(1) Gold palladium platinum nano enzyme bactericide for killing methicillin-resistant staphylococcus aureus
The methicillin-resistant staphylococcus aureus strain is inoculated into LB broth, and then cultured for 4 to 6 hours in a 37 ℃ incubator. The logarithmic growth phase was measured with a micro-UV spectrophotometer, and the optical density value at 600nm (OD 600 ) When the concentration of the strain is 0.5, the strain is diluted 50 times by LB broth culture solution, and the concentration of the strain is 2 multiplied by 10 6 CFU/mL, ready for use.
The grouping is set as follows:
0.9% nacl group: 100 mu L of methicillin-resistant staphylococcus aureus bacterial liquid plus 300 mu L of 0.9% NaCl;
acetic acid/sodium acetate buffer group: 100. Mu.L of methicillin-resistant staphylococcus aureus bacterial liquid+200. Mu.L of 0.9% NaCl+100. Mu.L of acetic acid/sodium acetate buffer;
4% polyethylene glycol group: 100. Mu.L of methicillin-resistant staphylococcus aureus bacterial liquid+100. Mu.L of 0.9% NaCl+100. Mu.L of acetic acid/sodium acetate buffer+100. Mu.L of 4% polyethylene glycol;
vancomycin group: 100 mu L of methicillin-resistant staphylococcus aureus bacterial liquid+200 mu L of 0.9 percent NaCl+100 mu L of vancomycin;
gold palladium platinum nano enzyme group: 100. Mu.L of methicillin-resistant Staphylococcus aureus solution+100. Mu.L of 0.9% NaCl+100. Mu.L of acetic acid/sodium acetate buffer+100. Mu.L of gold palladium platinum nano-enzyme solution (final concentrations: 7.81, 12.50, 15.63 and 31.25. Mu.g/mL).
Sequentially adding the test solutions into the tubes according to the above groups, incubating for 2 hours in a water bath at 37 ℃, transferring 10 mu L of the test solutions onto an agar culture dish, culturing in a biochemical incubator at 37 ℃, and observing the growth condition of colonies.
(2) Observing the bacterial morphology of the gold palladium platinum nano enzyme bactericide after the effect of methicillin-resistant staphylococcus aureus by a transmission electron microscope
Culturing methicillin-resistant staphylococcus aureus bacterial liquid to OD according to the method 600 And (5) standby.
The grouping is set as follows:
0.9% nacl group: 2mL of methicillin-resistant staphylococcus aureus bacterial liquid+2 mL of 0.9% NaCl;
gold palladium platinum nano enzyme group: 2mL of methicillin-resistant staphylococcus aureus bacterial liquid and 2mL of gold palladium platinum nano enzyme solution (final concentration: 15.63 mug/mL).
Sequentially adding the test solutions into the tube according to the above groups, incubating for 2 hours in a water bath at 37 ℃, and centrifuging 4000g of the test solution for 6 minutes, and washing the test solution for 3 times with phosphate buffer. Sufficient 2.5% glutaraldehyde was added for fixation, stored at 4℃overnight and the bacterial morphology was observed under transmission electron microscopy.
(3) Killing other pathogenic bacteria by gold palladium platinum nano enzyme
Culturing 5 common pathogenic bacteria (enterococcus faecalis, staphylococcus aureus, escherichia coli, klebsiella pneumoniae, acinetobacter baumannii and Pseudomonas aeruginosa) to OD according to the above method 600 At 0.5, the bacterial liquid was diluted 50-fold with LB broth, and the concentration of the bacterial liquid was 2X 10 6 CFU/mL, ready for use.
The grouping is set as follows:
0.9% nacl group: 100. Mu.L of bacterial liquid+300. Mu.L of 0.9% NaCl;
gold palladium platinum nano enzyme group: 100. Mu.L of bacterial liquid+100. Mu.L of 0.9% NaCl+100. Mu.L of acetic acid/sodium acetate buffer+100. Mu.L of gold palladium platinum nano-enzyme solution (final concentration: 15.63. Mu.g/mL).
Sequentially adding the test solutions into the tubes according to the above groups, incubating for 2 hours in a water bath at 37 ℃, transferring 10 mu L of the test solutions onto an agar culture dish, culturing in a biochemical incubator at 37 ℃, and observing the growth condition of colonies.
2. Analysis of experimental results
As shown in FIG. 5, when the concentration of the golden palladium platinum nano enzyme is 12.50 mug/mL, only a very small number of colonies grow on the agar plate, which is equivalent to the colony number of the antibiotic vancomycin flat plate, but when the concentration of the golden palladium platinum nano enzyme is 15.63 mug/mL, the golden palladium platinum nano enzyme bactericide is proved to have excellent performance of killing methicillin-resistant staphylococcus aureus.
As shown in FIG. 6, under the action of the gold-palladium-platinum nano-enzyme bactericide, the bacterial cell structure is destroyed, the bacterial content leaks, the bacteria die, and the bacteria in the 0.9% NaCl group do not exist.
As shown in FIG. 7, under the effect that the concentration of the golden palladium platinum nano enzyme is 15.63 mug/mL, all agar plates grow aseptically, which shows that the golden palladium platinum nano enzyme has excellent effect of killing enterococcus faecalis, staphylococcus aureus, escherichia coli, klebsiella pneumoniae, acinetobacter baumannii and pseudomonas aeruginosa, and also shows that the golden palladium platinum nano enzyme has excellent broad-spectrum sterilization performance. The reason why the gold-palladium-platinum nano enzyme bactericide can effectively kill pathogenic bacteria is that the active oxygen with strong oxidability is generated by catalyzing oxygen or hydrogen peroxide through the oxidase-like activity and the peroxidase-like activity, and the active oxygen can destroy active ingredients of bacteria, so that the bacterial morphology is changed drastically and finally the bacteria die.
Example 4
This example evaluates cytotoxicity of the gold palladium platinum nano enzyme bactericide with a size of 46.43nm prepared in example 1, and is implemented as follows:
1. experimental method
In order to evaluate the cytotoxicity of the gold palladium platinum nano enzyme bactericide, the present example was verified by a hemolysis experiment. 1mL of whole blood of healthy people is taken, 10mL of 0.9% NaCl is added for suspension, centrifugation is carried out at 6000rpm for 5min, supernatant fluid is removed, and repeated washing is carried out on the supernatant fluid by using 0.9% NaCl, so that the supernatant fluid does not appear red or slightly yellow. The lower layer of erythrocytes was resuspended with 4 volumes of 0.9% nacl to give a suspension of erythrocytes.
The grouping is set as follows:
gold palladium platinum nano enzyme group: gold palladium platinum nano enzyme bactericide (final concentration 8.93, 10.42, 12.50, 15.63, 20.83 and 31.25 mug/mL); negative control group: 0.9% NaCl; positive control group：H 2 O。
500 mu L of gold-palladium-platinum nano enzyme solution with different concentrations and H 2 O and 0.9% NaCl are respectively mixed with 50 mu L of erythrocyte suspension, and the mixture is placed in a water bath kettle at 37 ℃ for incubation for 1h. After the incubation was completed, the supernatant was centrifuged at 6000rpm for 5 minutes, the color of each group of supernatant was observed, the ultraviolet absorbance at 450 to 700nm was measured, and the hemolysis ratio was calculated according to the following formula.
Hemolysis ratio (%) = [ (a) 1 -A 2 )/(A 3 -A 2 )]×100％
Wherein A is 1 Is absorbance of gold palladium platinum nano enzyme group, A 2 Absorbance of negative control group (0.9% NaCl), A 3 Is a positive control group (H) 2 O) absorbance.
2. Analysis of experimental results
As shown in FIG. 8, the hemolysis rate was lower than 5% at the concentration of the gold-palladium-platinum nano-enzyme bactericide of less than 31.25. Mu.g/mL, and no obvious hemolysis phenomenon was shown, which suggests that the gold-palladium-platinum nano-enzyme bactericide of less than 31.25. Mu.g/mL did not cause serious hemolysis, and had good biosafety.
Example 5
The embodiment adopts the gold palladium platinum nano enzyme bactericide with the size of 46.43nm prepared in the embodiment 1 to treat wound infection of mice, and the animal experiment proves that the in-vivo sterilization performance of the bactericide is as follows:
1. experimental method
To evaluate the in vivo bactericidal effect of the gold palladium platinum nanoenzyme bactericides, female Balb/c mice (about 4-5 weeks, balb/c, about 14g, 3 per group) were used to establish a model of wound infection in mice. The mice were weighed, labeled, anesthetized with diethyl ether, shaved, and a circular skin wound of about 6mm diameter was produced on the back of each mouse with a punch, and 80. Mu.L of a strain containing methicillin-resistant Staphylococcus aureus (10 8 CFU/mL) was placed on the wound for infection. 24h after infection, mice were randomized into 3 groups and group treatment was set up as follows:
(1) Phosphate buffer group: the therapeutic agent was phosphate buffer (0.01M, pH 7.4) at a dose of 50. Mu.L per site;
(2) Vancomycin group: the therapeutic agent was vancomycin (4 mg/mL) at a dose of 50. Mu.L per site;
(3) Gold palladium platinum nano enzyme group: the therapeutic agent was a gold palladium platinum nanoenzyme solution (15.63 μg/mL) at a dose of 50 μl per site.
Wound photographs of each group were taken at each treatment time point. The course of treatment starts at 1d, once a day.
2. Analysis of experimental results
As shown in fig. 9, the experimental result proves that compared with the antibiotic vancomycin, the golden palladium platinum nano enzyme antibacterial agent has the same degree of bactericidal effect and can better and faster promote the wound healing of mice.
The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the invention. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is intended that all equivalent modifications and variations of the invention be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.
Claims (10)
1. The gold-palladium-platinum nano enzyme bactericide is characterized by comprising gold-palladium-platinum nano enzyme, wherein the concentration of the gold-palladium-platinum nano enzyme in the bactericide is less than or equal to 20.83 mug/mL; the bactericide has broad-spectrum bactericidal performance.
2. The gold-palladium-platinum nano-enzyme bactericide according to claim 1, which is characterized in that: the concentration of the gold-palladium-platinum nano enzyme is 7.0-20.83 mug/mL.
3. The gold-palladium-platinum nano-enzyme bactericide according to claim 1, which is characterized in that: the gold palladium platinum nano-enzyme is in a spherical outline, and the average size is 40-110 nm.
4. The gold-palladium-platinum nano-enzyme bactericide according to claim 1, which is characterized in that: the bactericide further comprises a solvent selected from at least one of water, acetic acid/sodium acetate buffer and phosphate buffer.
5. A method for preparing a gold-palladium-platinum nano-enzyme bactericide according to any one of claims 1 to 4, which is characterized by comprising the following steps:
b1, synthesizing gold seeds based on a seed-mediated growth method, and preparing a wine-red gold seed solution;
b2, mixing the gold seed solution with a hexadecyl trimethyl ammonium chloride solution, a palladium salt solution, a platinum salt solution and a reducing agent solution, and standing and aging at 50-70 ℃; centrifuging and collecting a product after aging is finished, and washing the product to obtain the gold-palladium-platinum nano enzyme;
and B3, adding the gold-palladium-platinum nano enzyme into a solvent to prepare the bactericide.
6. The method for preparing the gold-palladium-platinum nano-enzyme bactericide according to claim 5, which is characterized in that: the step B1 is based on a seed-mediated growth method to synthesize gold seeds, and the process of preparing the gold seed solution with the color of reddish wine comprises the following steps:
b11, mixing a gold salt solution and a cetyltrimethylammonium bromide solution, adding an ice-cold reducing agent solution, and immediately turning the solution into brown to obtain an initial small gold seed solution;
and B12, mixing a gold salt solution, a cetyltrimethylammonium chloride solution and a reducing agent solution, adding an initial small gold seed solution, standing, centrifugally collecting a product, and dispersing the product in water to obtain a wine-red gold seed solution.
7. The method for preparing the gold-palladium-platinum nano-enzyme bactericide according to claim 6, which is characterized in that: in the steps B11 and B12, the process conditions are selected from at least one of the following (1) to (6):
(1) in the steps B11 and B12, the reducing agent is at least one selected from sodium borohydride, ascorbic acid and sodium citrate;
(2) in the step B11, the molar ratio of gold ions in the gold salt solution, the cetyltrimethylammonium bromide solution and the reducing agent is 2-3:800-1200:4-8;
(3) in the step B11, the ice-cold reducing agent solution is a reducing agent solution at 4 ℃ or passing through an ice-water bath;
(4) in the step B12, the molar ratio of gold ions in the gold salt solution, the cetyltrimethylammonium bromide solution and the reducing agent is 1-3:600-1000:200-400;
(5) in the step B12, standing time is 20-40 min;
(6) in the step B12, the centrifugation time is 60-80 min, and the rotating speed is 14000-20000 rpm.
8. The method for preparing the gold-palladium-platinum nano-enzyme bactericide according to claim 5, which is characterized in that: in the steps B2 and B3, the process conditions are selected from the following (7) to (7)At least one of:
(7) in the step B2, the molar ratio of the gold seeds, the cetyl trimethyl ammonium chloride solution, the palladium ions in the palladium salt solution and the platinum ions in the platinum salt solution to the reducing agent is 0.05-1.2:30-50:1:1:80-120;
(8) in the step B2, the ageing time is 2-4 hours;
(9) in the step B2, the centrifugation time is 8-15 min, and the rotating speed is 14000-20000 rpm;
in the step B2, the reducing agent is at least one selected from sodium borohydride, ascorbic acid and sodium citrate;
in the step B3, the solvent is at least one selected from water, acetic acid/sodium acetate buffer and phosphate buffer.
9. Use of a gold-palladium-platinum nano-enzyme bactericide according to any one of claims 1 to 4 and/or a gold-palladium-platinum nano-enzyme bactericide prepared according to the method of any one of claims 5 to 8 as or in the preparation of a medicament and/or bactericide for the treatment of a disease caused by bacterial infection.
10. The use according to claim 9, characterized in that: the medicine and/or bactericide for treating diseases caused by bacterial infection is applied to in vivo sterilization.
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