CN113427015B - Preparation method and application of novel silver nano material AgNFs - Google Patents
Preparation method and application of novel silver nano material AgNFs Download PDFInfo
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- CN113427015B CN113427015B CN202110678518.0A CN202110678518A CN113427015B CN 113427015 B CN113427015 B CN 113427015B CN 202110678518 A CN202110678518 A CN 202110678518A CN 113427015 B CN113427015 B CN 113427015B
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B22—CASTING; POWDER METALLURGY
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- B22F9/00—Making metallic powder or suspensions thereof
- B22F9/16—Making metallic powder or suspensions thereof using chemical processes
- B22F9/18—Making metallic powder or suspensions thereof using chemical processes with reduction of metal compounds
- B22F9/24—Making metallic powder or suspensions thereof using chemical processes with reduction of metal compounds starting from liquid metal compounds, e.g. solutions
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
- A61K31/7036—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin having at least one amino group directly attached to the carbocyclic ring, e.g. streptomycin, gentamycin, amikacin, validamycin, fortimicins
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
- A61K33/24—Heavy metals; Compounds thereof
- A61K33/38—Silver; Compounds thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/02—Inorganic compounds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- 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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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y40/00—Manufacture or treatment of nanostructures
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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
- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
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- 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
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- 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
Abstract
The invention discloses a preparation method of novel silver nano material AgNFs, which comprises the following steps: (1) Weighing BSA, adding the BSA into water, stirring, and putting the BSA into an oil bath pan for stirring and preheating; (2) adding a silver ion solution after preheating is finished, and reacting; (3) Finally, dropwise adding an ascorbic acid aqueous solution, and continuing the reaction; (4) After the reaction is finished, the silver nano material AgNFs is obtained by cleaning, is dispersed and stored by ultrapure water, and is dispersed by ultrasonic before use. The AgNFs of the silver nano material has a simple synthetic path, is environment-friendly, has uniform material size, and has good dispersibility and stability; specifically, based on the enrichment and loading of AgNFs on antibiotics, the concentration of local antibiotics is improved, the metabolic activity of drug-resistant bacteria is regulated, agNFs stimulate cell respiration, and the antibiotics are taken in a synergistic manner to play an antibacterial role by means of proton motility enhancement.
Description
Technical Field
The invention relates to the technical field of preparation and application of silver nano materials, in particular to novel silver nano materials AgNFs, a preparation method and application thereof.
Background
The problem of antibiotic resistance is a major issue related to global health, and it is estimated that antibiotic resistance in 2017 brings about 770 billion dollars in socio-economic burden on China, which is equivalent to 0.37% of the total annual domestic production value. Bacterial resistance is further increased by overuse and misuse of antibiotics. The resulting failure of antibiotic therapy seriously compromises public health safety.
With the continuous development of nanotechnology, researchers have been exploring the use of nanomaterials in the field of combating microbial infections. Since metallic silver itself has excellent antibacterial properties, silver nanomaterials are being developed for antibacterial practice. In the existing research, researchers have studied the antibacterial action of silver nano-materials by synthesizing silver nano-materials with different sizes and shapes, such as truncated triangular nano-plate, nanospheres, pentagonal rods, pyramids and the like. The silver nano material can cause cytoplasm leakage to inactivate by destroying the integrity of bacterial cell wall; in addition, silver ions released by the nano material have high affinity with thiol-containing enzyme or protein in bacteria, and after the silver ions are combined, the accumulation of active oxygen is triggered, so that the physiological function of cells is disturbed, and the cells are killed. In view of this, combining a silver nanomaterial with an antibiotic can fully exert the antibacterial abilities of the two components, and in the existing research, researchers mostly combine the nanomaterial with the antibiotic on a synthetic route, such as vancomycin and dendrimer polyamide to resist staphylococcus aureus, or reduce metallic silver to a nano form by using an amine group of ampicillin to kill bacteria.
The existing synthesis methods of silver nano materials are mostly chemical methods, sonochemical methods and photochemical methods, the steps are complicated, and if an organic reagent is introduced, the method is not environment-friendly; in addition, the existing silver nano material needs higher concentration if being used singly to kill bacteria, thereby increasing toxicity and causing unnecessary killing to body cells, but does not relate to killing to drug-resistant bacteria.
Therefore, those skilled in the art are dedicated to develop a silver nano material, which has mild synthesis method, is environment-friendly, has the function of supporting and enriching drugs, especially hydrophobic antibiotics such as norfloxacin to assist the sterilization thereof, and particularly relates to a silver nano material which can enhance the killing effect of antibiotics on drug-resistant bacteria and reduce the sterilization dosage by the cell wall rupture of the bacteria caused by the release of silver ions and the change of metabolic state.
Disclosure of Invention
In view of the above defects in the prior art, the technical problem to be solved by the present invention is to obtain a preparation method and an application of a silver nano material with a function of supporting and enriching a drug, especially a function of assisting the sterilization of a hydrophobic antibiotic such as norfloxacin.
In order to realize the aim, the invention provides a preparation method of novel silver nano material AgNFs, which comprises the following steps:
(1) Weighing BSA, adding the BSA into water, stirring, and putting the BSA into an oil bath pan for stirring and preheating;
(2) Adding silver ion solution after preheating, and reacting;
(3) Finally, dropwise adding an ascorbic acid aqueous solution, and continuing the reaction;
(4) After the reaction is finished, the silver nano material AgNFs is obtained by cleaning, dispersed and stored by ultrapure water, and dispersed by ultrasonic before use.
Furthermore, the AgNFs are petal-shaped, and the size of the AgNFs is 700-800 nm.
Further, in step (1), 50mg of the BSA was weighed and added to 9ml of the water and stirred, and then placed in the oil bath at 30-60 ℃ and stirred at about 1500rpm for 5 minutes.
Further, in the step (2), 10mM of the silver ion solution was added and the reaction was carried out for 30 minutes.
Further, 1ml of the ascorbic acid aqueous solution containing 50mg of ascorbic acid was added dropwise in step (3), and the reaction was continued for 30min.
Further, the ascorbic acid aqueous solution was added at a rate of 20. Mu.l/drop.
Further, in the step (4), centrifugation is carried out at 5000rpm for 25min, and washing is carried out twice.
Further, the silver nano material AgNFs in the step (4) is dispersed and stored in 5ml of ultrapure water.
The invention also provides application of the novel silver nano material AgNFs in loading antibiotics.
Further, the antibiotic is one or two of norfloxacin and streptomycin sulfate.
The material synthesis of the invention uses biological macromolecules (bovine serum albumin), which is not only environment-friendly, but also has no potential harm to organisms; the nano material has a unique secondary structure, has remarkable norfloxacin carrying capacity, and can reduce the dosage of the medicament by enriching the medicament; meanwhile, substances such as hydrogen peroxide generated by bacteria per se are utilized to etch the silver nano material, more silver ions are released, so that the dosage of the nano material is reduced, and the biological safety is ensured on the whole; the material is verified by in vitro experiments, and a large amount of medicines and silver ions enter thalli by loading medicines and destroying the integrity of bacteria, so that the aim of killing drug-resistant bacteria is fulfilled while the sterilization dose is reduced; provides a feasible solution for killing drug-resistant bacteria, solves the drug-resistant problem from the downstream of a drug-resistant mechanism, namely the energy metabolism, avoids the specificity of a drug-resistant target, and has the feasibility of killing quinolone-resistant escherichia coli in a broad spectrum.
The conception, specific structure and technical effects of the present invention will be further described in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention.
Drawings
FIG. 1 is a scanning electron microscope representation of silver nanomaterials AgNFs of a preferred embodiment of the invention;
FIG. 2 is an untreated bacterial morphology (blank control);
fig. 3 is the bacterial morphology after AgNFs treatment (AgNFs treatment group alone);
FIG. 4 shows the morphology of bacteria treated by AgNFs and norfloxacin (AgNFs and norfloxacin treatment group);
FIG. 5 is an oxford cup experiment verifying the antibacterial effect on resistance to 50 times norfloxacin MIC;
FIG. 6 is an oxford cup experiment verifying the antibacterial effect on 500 times streptomycin sulfate resistant MIC;
figure 7 is an oxford cup experiment demonstrating the antimicrobial effect against a 5-fold (norfloxacin + streptomycin sulfate combination) MIC resistance.
Detailed Description
The technical contents of the preferred embodiments of the present invention will be more clearly and easily understood by referring to the drawings attached to the specification. The present invention may be embodied in many different forms of embodiments and the scope of the invention is not limited to the embodiments set forth herein.
Example 1
Preparation of materials
50mg of BSA was weighed and added to 9ml of water, stirred at about 1500rpm in an oil bath at 30-60 ℃ for 5 minutes, and after completion of preheating, a 10mM silver ion solution was added thereto, and reacted for 30 minutes. Finally, 1ml of ascorbic acid aqueous solution containing 50mg of ascorbic acid was added dropwise, the reaction was continued for 30min, centrifuged at 5000rpm for 25min, and washed twice. The resulting mixture was stored in 5ml of ultrapure water and dispersed by ultrasonic waves before use.
Example 2
Antibacterial experiments
1. The antibacterial effect of AgNFs or norfloxacin used alone and AgNFs and norfloxacin used in combination on norfloxacin-resistant escherichia coli is evaluated by a growth curve determination method, and the result shows that the dosages of AgNFs and norfloxacin used when the combination is used to achieve the bactericidal effect are respectively reduced by 50% and 98% compared with the dosages when the AgNFs or norfloxacin is used alone. The shapes of the bacteria treated by the blank control group, the single AgNFs treatment group and the AgNFs and norfloxacin treatment group are shown in fig. 2, fig. 3 and fig. 4.
2. The antibacterial effect of AgNFs or streptomycin sulfate used alone and AgNFs and streptomycin sulfate used in combination on streptomycin sulfate-resistant Escherichia coli was evaluated by a growth curve assay, and the results showed that the dosages of AgNFs and streptomycin sulfate used in combination to achieve the bactericidal effect were reduced by 80% and 98%, respectively, compared to when used alone.
3. The antibacterial effect of AgNFs or (norfloxacin + streptomycin sulfate) drug pairs used alone and AgNFs and (norfloxacin + streptomycin sulfate) drug pairs used in combination on streptomycin sulfate-resistant Escherichia coli was evaluated by a growth curve assay, and the results show that the dosages of AgNFs and drug pairs used in combination to achieve the bactericidal effect were reduced by 90% and 50%, respectively, compared with the dosages used alone.
In addition, the results of the oxford cup experiments on the three drug-resistant bacteria are shown in fig. 5, fig. 6 and fig. 7, the inhibition zone shows the generation of inhibition, and 1,2,3 and 4 in the figures sequentially show a blank control group, an antibiotic treatment group, an AgNFs treatment group and an AgNFs + antibiotic treatment group. Fig. 5 to 7 are drug-resistant e.coli at three levels of resistance, where strain I: tolerating 50 times of norfloxacin MIC; strain II: tolerating 500 times streptomycin sulfate MIC; strain III: tolerates 5-fold (norfloxacin + streptomycin sulfate combination) MIC.
The invention designs a novel silver nano material which takes biomacromolecules (bovine serum albumin) as raw materials and is rapidly synthesized under mild conditions (30-60 ℃). The material has uniform size, good dispersibility and good stability. In addition, the material can be seen to have a fine secondary structure through a high-resolution photograph (figure 1) of an electron microscope, and meanwhile, the material is full of nano silver particles, so that the high specific surface area can realize the loading of the drugs, particularly the antibiotics. Can be used as a carrier of antibiotics such as (norfloxacin) to realize the enrichment of drug concentration, and simultaneously release silver ions to enhance the killing of the antibiotics to drug-resistant escherichia coli. After the nano material is combined with norfloxacin, the norfloxacin loading rate reaches nearly 100 percent; the minimum inhibitory concentration of the nano material and the antibiotic is respectively reduced by 50 percent and 98 percent, the dosage is reduced, the toxic effect of the nano material and the drug is reduced, and the biological safety is ensured while the drug-resistant bacteria killing is realized.
The novel silver nano material (AgNFs) synthesized by the method has obvious carrying capacity (nearly 100%) on quinolone antibiotics such as norfloxacin, and meanwhile, positive charges carried by the silver nano material are mutually adsorbed after contacting bacterial cells with negative charges, so that the high-concentration antibiotics are effectively enriched near thalli; in addition, the silver nano material damages the integrity of cell walls, provides conditions for silver ions and drug molecules to enter the interior of bacteria, and further enhances the killing effect of the silver ions and the drug molecules on important proteins and DNA of the bacteria. In order to form a petal-shaped secondary structure with high specific surface area and realize good loading and enrichment of antibiotics, ascorbic acid is slowly added dropwise (20 mu l/drop) as much as possible to perform reduction reaction in the synthesis step of the silver nano material.
In-vitro experiments prove that the novel silver nano material can kill single high-drug-resistance bacteria and multiple drug-resistance bacteria while reducing the dosage of antibiotics. The specific principle includes, in addition to the above-mentioned increase of the local antibiotic concentration: a large amount of hydrogen peroxide generated by bacteria under the stimulation of antibiotics is used for etching the silver nano material, more silver ions are generated to promote the bacteria to absorb the antibiotics, the discharge effect of drug-resistant bacteria to the antibiotics is reversed, and therefore positive feedback circulation of mutual promotion of AgNFs and the antibiotics is generated, and the drug-resistant bacteria are killed.
The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions that can be obtained by a person skilled in the art through logical analysis, reasoning or limited experiments based on the prior art according to the concepts of the present invention should be within the scope of protection determined by the claims.
Claims (4)
1. The application of the silver nanomaterial AgNFs in antibiotic loading is characterized by comprising the following steps:
(1) Weighing 50mg BSA, adding into 9ml water, stirring, putting into an oil bath pan at the temperature of 30-60 ℃, stirring at 1500rpm for 5 minutes, and preheating;
(2) After preheating, adding 10mM silver ion solution, and reacting for 30 minutes;
(3) Adding 1ml of ascorbic acid aqueous solution containing 50mg of ascorbic acid dropwise at the speed of 20 mul/drop, and continuing the reaction for 30min;
(4) After the reaction is finished, cleaning to obtain silver nano material AgNFs, wherein the silver nano material AgNFs are petal-shaped, the size of the silver nano material AgNFs is 700-800nm, dispersing and storing the silver nano material AgNFs by using ultrapure water, and dispersing the silver nano material AgNFs by using ultrasonic waves before use;
(5) And carrying antibiotics on the nano material AgNFs.
2. The application of the silver nanomaterial AgNFs to the loading of antibiotics as claimed in claim 1, wherein in step (4), centrifugation is performed at 5000rpm for 25min, and washing is performed twice.
3. The application of the silver nano material AgNFs in carrying antibiotics in claim 1, wherein the silver nano material AgNFs is stored in 5ml of ultrapure water in a dispersing way in the step (4).
4. The use of the silver nanomaterials AgNFs according to claim 1 in the loading of antibiotics, wherein the antibiotics are one or both of norfloxacin and streptomycin sulphate.
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| CN110186969A (en) * | 2019-05-29 | 2019-08-30 | 上海交通大学 | Based on AgNFs-Pt nanocomposite without enzyme electrochemical hydrogen peroxide detector and preparation |
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| WO2005120173A2 (en) * | 2004-05-12 | 2005-12-22 | Kishore Madhukar Paknikar | Anti-microbial activity of biologically stabilized silver nano particles |
| CN100502890C (en) * | 2006-03-02 | 2009-06-24 | 同济大学 | Amoxicillin antibacterial agent containing nano silver, and its preparing method |
| EP2950804B1 (en) * | 2013-01-30 | 2017-12-06 | Univerzita Palackého v Olomouci | Antibiotic preparation and use thereof |
| CN104690288B (en) * | 2014-11-27 | 2017-03-22 | 南京邮电大学 | Preparation method of flower-like gold nanoparticles and application of flower-like gold nanoparticles serving as SERS (surface enhanced Raman scattering) enhanced substrates |
| CN105998062A (en) * | 2016-04-14 | 2016-10-12 | 上海大学 | Antibacterial combination containing nano silver and kanamycin |
| US11304974B2 (en) * | 2017-04-10 | 2022-04-19 | Laboratorios Enosan, S.L. | Nanosystems comprising silver and antibiotics and their use for the treatment of bacterial infections |
| CN107127355B (en) * | 2017-07-04 | 2019-07-19 | 广东工业大学 | A kind of flower-shaped silver nanoparticle ball, preparation method and its application |
| CN110076349B (en) * | 2019-06-13 | 2022-02-18 | 江苏汇诚医疗科技有限公司 | Green preparation method of petal-shaped silver nanosheets |
| US10751802B1 (en) * | 2019-10-03 | 2020-08-25 | King Saud University | Method of producing silver nanoparticles using red sand |
| CN111673090B (en) * | 2020-06-09 | 2022-12-20 | 中北大学 | Preparation method of flower-like silver by using gallic acid as structure-directing agent |
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