CN115770256A - Application of silver nano enzyme assembly/antibiotic in preparation of combined medicine for treating drug-resistant infectious diseases - Google Patents
Application of silver nano enzyme assembly/antibiotic in preparation of combined medicine for treating drug-resistant infectious diseases Download PDFInfo
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- CN115770256A CN115770256A CN202211413003.9A CN202211413003A CN115770256A CN 115770256 A CN115770256 A CN 115770256A CN 202211413003 A CN202211413003 A CN 202211413003A CN 115770256 A CN115770256 A CN 115770256A
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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
- 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
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Abstract
The invention discloses an application of a silver nanoenzyme assembly in preparation of a medicine for treating drug-resistant infectious diseases by cooperating with antibiotics, wherein the silver nanoenzyme assembly is composed of ultra-small silver nanoenzyme, an infection microenvironment responsive polymeric ligand and an amphiphilic surfactant. The silver nanoenzyme assembly provided by the invention can specifically exert the activity of similar oxidase in an infection microenvironment to destroy a bacterial biofilm, enter bacterial cells to exert the activity of similar thiol oxidase to catalyze thiol in ribosomal protein cysteine residues to be oxidized into sulfonic acid, and interfere the biosynthesis function of ribosome, so that the secretion of drug-resistant substances is inhibited, and the curative effect of antibiotics is enhanced.
Description
Technical Field
The invention relates to application of a nano biological material and antibiotic combined medicine, in particular to application of a silver nano enzyme assembly/antibiotic in preparation of a combined medicine for treating drug-resistant infectious diseases.
Background
Resistant bacterial infections have become an important public health problem worldwide. Drug-resistant bacterial infections are reported to be expected to cause 1000 million deaths per year by 2050, and treatment costs of up to $ 100 trillion, pose a significant health threat and economic burden. The reason for this problem is that the drug-resistant bacteria escape from the antibiotic efficacy by reducing the antibiotic concentration in the bacteria, secreting antibiotic inactivating enzymes, modifying antibiotic targets, etc., and the efficacy of most antibiotics is threatened at present. However, no antibiotics with novel mechanisms have been approved for the market in the last 30 years, which further increases the treatment burden of drug-resistant bacterial infections, and therefore, the preparation of novel combination drugs based on the existing antibiotics is a key means for overcoming bacterial drug resistance.
At present, small molecule inhibitors can enhance the action of antibiotics by interfering with the action of antibiotic inactivating enzymes, destroying the stability of bacterial membranes, or inhibiting antibiotic efflux pumps on bacterial membranes, so that antibiotics can be used to fight against drug-resistant bacteria. For example, chinese patent publication No. CN107753470 discloses a small molecule inhibitor of polymyxin drug-resistant protein and its application. The invention discovers for the first time that ethanolamine serving as a small molecular inhibitor can inhibit the drug resistance of polymyxin, inhibit the activity of MCR-1 protein and inhibit the activity of phosphotransferase with a structure similar to that of MCR-1, and the ethanolamine serving as the small molecular inhibitor and the polymyxin are combined to enable the polymyxin to play a role better, so that a more effective application space is provided for the application of the polymyxin, the ethanolamine has very important application value in the pharmaceutical field and the animal breeding field, and has great significance today for the wide existence of the drug resistance.
However, bacteria secrete extracellular matrix and spontaneously form biofilms after colonization in the organism, providing a physical barrier that protects the bacteria from small molecule antibiotics and inhibitors; meanwhile, the biosynthesis and secretion actions mediated by ribosome biogenesis in bacterial cells further support bacterial adhesion, the continuous formation of biological membranes and the generation of substances (such as antibiotic inactivated enzymes) related to bacterial drug resistance, and provide a chemical barrier. The extracellular and intracellular vital activities of these bacteria cooperate to form a microenvironment favorable for the growth of drug-resistant bacteria, i.e., an infection microenvironment, which hinders the practical application effects of antibiotics and drug-resistant inhibitors. Therefore, the development of drugs that can regulate the infection microenvironment, especially those that can disrupt bacterial biofilms, and inhibit bacterial ribosome function, would hopefully disrupt bacterial resistance barriers for the preparation of novel antibacterial drugs complexed with antibiotics to overcome drug-resistant bacterial infections.
Disclosure of Invention
The invention aims to provide an application of a silver nanoenzyme assembly/antibiotic in preparation of a combined drug for treating drug-resistant infectious diseases, wherein the silver nanoenzyme assembly can specifically exert the activity of a similar oxidase in an infection microenvironment to destroy a bacterial biomembrane, enter bacterial cells to exert the activity of the similar thiol oxidase to catalyze the oxidation of thiol in cysteine residues of ribosomal protein into sulfonic acid, and interfere with the biosynthesis function of ribosome, so that the secretion of drug-resistant substances is inhibited, and the curative effect of the antibiotic is enhanced.
The invention provides the following technical scheme:
the application of the silver nanoenzyme assembly/antibiotic in the preparation of the combined medicament for treating the drug-resistant infectious diseases comprises the silver nanoenzyme assembly and the antibiotic, wherein the silver nanoenzyme assembly is composed of ultra-small silver nanoenzyme, infection microenvironment responsive macromolecular ligand and amphiphilic surfactant.
The silver nano enzyme assembly has the activity of similar oxidase and the activity of similar thiol oxidase: after being disassembled and assembled in an infection microenvironment, the activity of the similar oxidase is exerted, and active oxygen (ROS) generated by catalysis can destroy a bacterial biomembrane and enhance the penetration of antibiotics; silver nanoenzyme enters bacterial cells to play a thiol-like oxidase activity, can be combined with and oxidize a sulfhydryl/disulfide bond on cysteine on ribosome through a silver-sulfur bond, and is oxidized into sulfonic acid (the thiol in the cysteine residue of ribosomal protein can be catalyzed and oxidized into the sulfonic acid), so that the secondary structure of ribosomal protein can be destroyed, the protein synthesis function of ribosome can be inhibited (the biosynthesis function of ribosome can be interfered), the secretion of drug-resistant substances can be inhibited, and the curative effect of antibiotics can be enhanced. The function of the silver nanoenzyme can effectively enhance the penetration effect and bactericidal activity of antibiotics in the combined medicament, so that the combined medicament can generate excellent treatment effect on infectious diseases.
The preparation method of the silver nano enzyme assembly comprises the following steps:
1) Preparing the ultra-small silver nano enzyme: preparing ultra-small silver nanoenzyme by a thermal decomposition method by taking silver nitrate as a precursor, oleylamine as a reducing agent and oleic acid as a solvent;
2) Preparing a silver nano enzyme assembly: mixing the ultra-small silver nanoenzyme and the infection microenvironment responsive macromolecular ligand in an organic solvent, slowly dropping the mixture into phosphate buffer solution dissolved with amphiphilic surfactant in water bath ultrasound, fully emulsifying, and volatilizing to remove the organic solvent to obtain the silver nanoenzyme assembly.
And mixing the silver nano enzyme assembly and the antibiotic in a phosphate buffer solution to obtain the silver nano enzyme assembly/antibiotic combined drug.
In the step 1), the mass ratio of the silver nitrate to the oleylamine is 1:2.9-5.9, and the heating rate is 1-10 ℃/min.
The size of the silver nanoenzyme can be controlled by controlling the temperature rise rate, when the ratio of silver nitrate: oleylamine (mass ratio) is 1:2.9, obtaining the ultra-small silver nano enzyme with the particle size of 3-5nm when the heating rate is 1 ℃/minute.
In step 2), the responsive group in the infection microenvironment-responsive polymeric ligand comprises one or more of gluconic acid, imidazole, hyaluronic acid, poly (epsilon-caprolactone), polyphosphate, gelatinase-cleavable peptide;
the amphiphilic surfactant is selected from one or more of poloxamer, polyethylene glycol-polystyrene and polyethylene glycol-polylactic acid.
The size of the ultra-small silver nanoenzyme is 3-5nm, the hydrated grain size of a silver nanoenzyme assembly obtained by assembling an infection microenvironment responsive macromolecular ligand and an amphiphilic surfactant is 200nm, and the hydrated grain size of the silver nanoenzyme assembly obtained by disassembly is 10-20nm.
The hydrated grain size of the silver nanoenzyme assembly is about 200nm in an assembled state, and an infection microenvironment responsive polymer ligand in the silver nanoenzyme assembly can generate structural change in response to microenvironment characteristics, so that the silver nanoenzyme assembly is converted into a dispersed state from the assembled state, the hydrated grain size of the silver nanoenzyme assembly is reduced to 10-20nm, the specific surface area of the silver nanoenzyme is increased, and the activity of the oxidase-like enzyme is enhanced. In the oxidation catalysis process, oxygen molecules are combined on the surface of the silver nanoenzyme, ROS are generated through catalysis, and oxygen single atoms are formed on the surface. The silver nanoenzyme carrying oxygen monoatomic atoms on the surface plays the activity of thiol-like oxidase after combining thiol groups, and can oxidize the thiol groups or disulfide bonds into sulfonic acid.
Preferably, the antibiotic is ampicillin, penicillin and streptomycin.
Preferably, the mass ratio of the silver nanoenzyme assembly to the antibiotic in the combined medicament is 8. The silver nano enzyme assembly and the antibiotic have optimal synergistic effect under the proportion, so that the ratio of the silver nano enzyme assembly to the antibiotic is 8:1 is effective in treating drug-resistant bacterial infections.
The silver nanoenzyme assembly provided by the invention can be responsively disassembled in a methicillin-resistant staphylococcus aureus biomembrane, deeply permeates into the biomembrane and catalyzes generation of ROS, and the biomembrane is oxidized and damaged; further, the disassembled silver nanoenzyme enters bacteria, combines cysteine on a ribosome and catalyzes sulfonation of thiol groups, destroys a protein secondary structure so as to interfere the function of the ribosome, inhibits synthesis and secretion of substances such as bacterial membrane protein, antibiotic inactivated enzyme, extracellular matrix and the like, finally reverses bacterial drug resistance, and enhances permeability and bactericidal activity of antibiotics in the combined drug.
Specifically, the silver nanoenzyme/antibiotic combined drug can effectively kill drug-resistant bacteria and remove a biological membrane, enhances the penetration of antibiotics by destroying the biological membrane, reduces the secretion level of antibiotic inactivated enzyme and extracellular matrix by inhibiting the ribosome biosynthesis function, reverses the drug resistance of the drug-resistant bacteria, and has a synergistic enhanced curative effect compared with the single treatment of the antibiotics with the same concentration or the silver nanoenzyme with the same concentration.
Meanwhile, the silver nanoenzyme assembly provided by the invention can inhibit the evolution of the drug resistance of bacteria to antibiotics by inhibiting the secretion of drug-resistant substances such as extracellular matrix and the like.
The silver nanoenzyme assembly has similar oxidase activity and similar thiol oxidase activity, can catalyze the generation of ROS, and can catalyze the oxidation of cysteine. After the silver nanoenzyme assembly enters an infection microenvironment, the silver nanoenzyme assembly can be disassembled and exerts oxidase-like activity, and ROS is generated to destroy a biological membrane barrier. When the silver nanoenzyme is further taken up by drug-resistant bacteria, the silver nanoenzyme can exert thiol-like oxidase activity, oxidize cysteine residues of ribosomal protein, and inhibit the ribosome biosynthesis function. Therefore, the silver nanoenzyme assembly not only can destroy a bacterial biological membrane to break a physical drug resistance barrier, but also can inhibit secretion of drug resistance related substances (including bacterial membrane proteins, antibiotic inactivated enzymes and extracellular matrix), so that the permeability and antibacterial activity of antibiotics in the combined drug are enhanced.
The mechanism of the silver nanoenzyme/antibiotic combined drug in the invention is as follows: the silver nano enzyme assembly can be disassembled in an infection microenvironment, and the monodisperse silver nano enzyme can efficiently permeate into a bacterial biomembrane to exert the activity of the similar oxidase to destroy the structure of the biomembrane. The monodisperse silver nanoenzyme is easy to be absorbed by drug-resistant bacteria and combined with cysteine residues in bacterial intracellular ribosome protein, and cysteine is oxidized to cause the damage of protein secondary structure, influence the ribosome biosynthesis function, amplify the antibiotic curative effect, so that the combined medicament presents good anti-infection curative effect in the proportion.
Compared with the prior art, the invention has the beneficial effects that:
(1) The silver nano enzyme assembly in the silver nano enzyme assembly/antibiotic combined drug consists of the ultra-small silver nano enzyme, and the ultra-small silver nano enzyme has ultra-small size and large specific surface area, so that the surface of the ultra-small silver nano enzyme has abundant catalytic sites, and high-efficiency enzyme-like catalytic reaction can be carried out.
(2) The silver nano enzyme assembly in the silver nano enzyme assembly/antibiotic combined drug has infection microenvironment responsiveness, and can be responsively disassembled in the microenvironment of an infected part, so that the specifically enhanced enzyme activity is exerted.
(3) The silver nano enzyme assembly/antibiotic combined drug can oxidize and damage the structure of a drug-resistant bacterial biomembrane and effectively permeate into the bacterial biomembrane, and in addition, the combined drug can reduce the secretion levels of extracellular matrix and antibiotic inactivated enzyme, thereby weakening the drug resistance of bacteria and improving the antibacterial activity of the bacteria.
Drawings
FIG. 1 is a Transmission Electron Microscope (TEM) image of ultra-small silver nanoenzymes having both an oxidase-like activity and a thiol-like oxidase activity prepared in example 1;
FIG. 2 is a TEM image of the silver nanoenzyme assemblies having oxidase-like activity and thiol-like oxidase activity prepared in example 1 at pH7.4 and pH 5.5;
fig. 3 is a dynamic light scattering particle size distribution diagram of the silver nanoenzyme assembly prepared in example 1 under different pH conditions.
Fig. 4 is a performance characterization of the silver nanoenzyme assembly prepared in example 1 exerting oxidase-like activity under acidic conditions.
FIG. 5 is a performance characterization of thiol-like oxidase activity exerted after disassembly of the silver nanoenzyme assemblies prepared in example 1;
FIG. 6 is a representation of the ability of the silver nanoenzyme assemblies prepared in example 1 to generate ROS in a simulated biofilm microenvironment, and to disrupt bacterial biofilms;
FIG. 7 shows the detection of the secretion levels of the bacterial membrane potential, extracellular matrix and antibiotic inactivating enzyme after the co-incubation of the drug-resistant bacteria and the silver nanoenzyme assembly.
FIG. 8 is a performance test of silver nanoenzyme assembly/antibiotic combination drug treatment of drug resistant bacterial biofilm infection using Propidium Iodide (PI) dye and dead cell nucleus (Syto Green) dye staining.
Fig. 9 is an evaluation of the effect of the silver nanoenzyme assembly/antibiotic combination drug prepared in example 1 on the treatment of bacterial biofilm infection of mouse skin.
Detailed Description
The silver nanoenzyme assembly provided by the invention can be responsively disassembled in an infection microenvironment, exerts the specific amplified oxidase-like activity, and generates ROS to destroy the structure of a bacterial biofilm; after entering bacterial cells, the silver nanoenzyme exerts mercaptan-like oxidase activity, oxidizes cysteine residues of ribosomal protein, inhibits the biosynthesis and secretion functions of ribosomes, reduces the secretion of drug-resistant related substances such as extracellular matrix, antibiotic inactivated enzyme and the like, and reverses the drug resistance of bacteria; the silver nanoenzyme assembly can be used as an antibacterial adjuvant to restore the sensitivity of drug-resistant bacteria to traditional antibiotics, and provides a strategy for the development of drugs capable of efficiently treating drug-resistant bacterial infection. The invention is further described with reference to the following specific embodiments and the accompanying drawings.
Example 1: synthesis and characterization of silver nanoenzyme assembly with oxidase-like activity and thiol-like oxidase activity
(1) Synthesizing ultra-small silver nanoenzyme:
mixing 170mg of silver nitrate with 0.5mL of oleylamine and 4.5mL of oleic acid, stirring for 1.5h at 70 ℃ under vacuum, heating the mixture to 180 ℃ at the speed of 1 ℃/min under the protection of argon, keeping the temperature for 2 min when the system temperature reaches 180 ℃, rapidly cooling, adding absolute ethyl alcohol for precipitation, centrifuging (8000rpm, 10min), discarding supernatant, collecting precipitate, centrifuging and washing for 3 times by using a mixed solution of chloroform and absolute ethyl alcohol to obtain the ultra-small silver nanoenzyme, and dispersing in chloroform for later use.
The result is shown in figure 1, the ultra-small silver nanoenzyme is characterized by the appearance by a transmission electron microscope, and the grain diameter is about 3-5nm.
(2) Synthesis of pH-responsive polymeric ligand:
0.2g of octadecylamine was weighed out and dissolved in 10mL of dichloromethane (CH) 2 Cl 2 ) In (1). Separately, 1.0g of L-benzyl aspartate-N-carboxyanhydride was weighed out, dissolved in 5mL of dimethyl sulfoxide (DMSO), and slowly added to the octadecylamine CH 2 Cl 2 In the solution, the reaction was stirred at 30 ℃ for 48h. After the reaction is finished, adding the reaction mixture into pre-cooled anhydrous ether, standing at 4 ℃ to separate out crystals, and centrifuging(3500rpm, 5 min), and centrifugally washing the solid with anhydrous ether for 3 times, and then placing the collected solid in a vacuum drying box to dry overnight at room temperature to obtain octadecylamine-poly (benzyl aspartate). 0.2g of dried octadecylamine-polyaspartic acid benzyl ester was weighed, dissolved in 5mL of DMSO, and 1mL of 1- (3-aminopropyl) imidazole was added thereto, and the reaction was stirred at room temperature for 12 hours. Subsequently, the reaction solution was added to 20mL of pre-cooled 0.1M hydrochloric acid (HCl), filled into an activated dialysis bag (molecular weight cut-off 1000 Da), and dialyzed against 0.01M HCl as a dialysis medium. And after the dialysis is finished, collecting liquid in the dialysis bag, and freeze-drying to finally obtain the pH-responsive polymer ligand octadecylamine-poly (aspartic acid-imidazole).
(3) Synthesizing a silver nano enzyme assembly:
weighing about 3mg of ultra-small silver nanoenzyme, and dispersing in 0.5mL of chloroform; weighing 5mg of octadecylamine-poly (aspartic acid-imidazole), dissolving in 0.1mL of anhydrous methanol, and uniformly mixing with a chloroform solution of the ultra-small silver nanoenzyme for later use. Separately, 10mg of the hydrophilic surfactant pluronic F127 was weighed and dissolved in 5mL of phosphate buffer (PBS, pH7.4, 10 mM). Slowly and dropwise adding the mixed solution of the ultra-small silver nanoenzyme and the octadecylamine-poly (aspartic acid-imidazole) into the pluronic F127 solution under the condition of water bath ultrasound, and continuously shaking the container to form a uniform emulsion. After the dropwise addition, the obtained uniform emulsion is placed in a water bath at 30 ℃, and the mixture is stirred for 30min in an open manner to completely volatilize the organic solvent, so that the silver nano-enzyme assembly is finally obtained.
The result is shown in fig. 2, the prepared silver nanoenzyme assembly is characterized in appearance by a transmission electron microscope, the silver nanoenzyme assembly keeps an assembled state under the condition of pH7.4, and is disassembled into a dispersed state under the condition of pH 5.5.
The result is shown in fig. 3, the hydrated particle size of the prepared silver nanoenzyme assembly is characterized by using a dynamic light scattering instrument, and the hydrated particle size of the silver nanoenzyme assembly is reduced along with the reduction of the pH, which indicates that the silver nanoenzyme assembly is disassembled along with the reduction of the pH.
The results are shown in fig. 4, and the oxidase-like activity of the prepared silver nanoenzyme assembly is characterized, and the absorbance of the o-phenylenediamine oxidation product during the co-incubation of the silver nanoenzyme assembly and the o-phenylenediamine is detected by an ultraviolet spectrophotometer in phosphate buffer solutions with pH7.4 and pH5.5, respectively, and the results show that the absorbance of the o-phenylenediamine oxidation product in the phosphate buffer solution with pH5.5 is stronger than that under the condition of pH7.4, which indicates that the silver nanoenzyme assembly exerts enhanced oxidase-like activity under the condition of pH 5.5.
The results are shown in fig. 5, and the thiol oxidase-like activity of the prepared silver nanoenzyme assembly is characterized, the silver nanoenzyme assembly is incubated in phosphate buffer solution with pH5.5 for disassembly, then incubated with L-cysteine under the conditions of phosphate buffer solution with pH7.4 and 37 ℃, and the characteristic peak change of cysteine thiol groups is detected by using an infrared spectrometer and a raman scattering spectrometer, so that the results show that the thiol characteristic peak is weakened, the sulfonic acid group characteristic peak is strengthened, and that the silver nanoenzyme can oxidize thiol to sulfonic acid.
Example 2: the silver nano enzyme assembly has the effects of destroying bacterial biomembranes and inhibiting the functions of ribosomes.
Preparation of the medicament: the silver nanoenzyme assembly prepared in example 1 was dispersed in a phosphate buffer solution of pH 7.4.
Establishing a drug-resistant bacteria biofilm model and a planktonic drug-resistant bacteria model: staphylococcus aureus was cultured in Tryptone Soy Broth (TSB) at pH5.5 and pH7.4, respectively, to simulate the growth environment and planktonic growth environment of drug-resistant bacteria in biofilms, respectively.
Group setting:
a. control group: drug-resistant bacteria biofilm/planktonic drug-resistant bacteria were given fresh tryptone soy broth.
b. Treatment group 1: adding Ag into the culture solution of drug-resistant bacteria biofilm/planktonic drug-resistant bacteria + The concentration was adjusted to 8. Mu.g/mL.
c. Treatment group 2: adding the silver nano enzyme assembly into the drug-resistant bacteria biomembrane/planktonic drug-resistant bacteria culture solution to ensure that the silver concentration is 8 mu g/mL.
Incubating the above three groups of culture solutions at 37 deg.C and 180rpm for 2h, adding ROS fluorescenceProbe 2',7' -dichloro-fluorescent yellow diacetate (DCFH-DA, 20. Mu.M) was incubated at 37 ℃ and 180rpm in the dark for 15min. Subsequently, the bacteria were collected by centrifugation (5000rpm, 5min,4 ℃) and washed 2 times with ultrapure water, finally resuspended with ultrapure water, and the fluorescence intensity therein (excitation wavelength of 488nm, emission wavelength of 525 nm) was measured by a fluorescence spectrophotometer while measuring OD by the same time 600 The total amount of bacteria was quantified and the mean fluorescence intensity was obtained as the ratio of fluorescence intensity to OD600. The intensity of ROS generation was evaluated by calculating the relative fluorescence intensity of each of the other groups with the average fluorescence intensity of the blank group under neutral conditions being 1.
Silver nanoenzyme assembly (8 mug/ml) or phosphate buffer and methicillin-resistant staphylococcus aureus (2 x 10) 5 CFU/ml) in a well plate, and culturing was continued at 37 ℃ for 24h in the presence/absence of catalase (28 units/ml), respectively. Subsequently, the plate was inverted to discard the supernatant, and washed 3 times with deionized water to wash out suspended bacteria. Then, the mixture was fixed with methanol, incubated at room temperature for 15min, and then removed by aspiration and air-dried. After fixation, 50. Mu.L of 0.05% crystal violet solution was added to each well for staining, and incubation was performed at room temperature for 5min. After dyeing, washing with deionized water for 3 times, and washing off redundant dyes. Subsequently, the well plate was left uncapped and placed in an oven (37 ℃ C.) for drying. After drying, 200. Mu.L of 33% acetic acid solution was added to each well and the dye was completely dissolved by gentle shaking for 15min. Finally, the absorption value of the crystal violet at 570nm is measured by a microplate reader, and the percentage of the total amount of the biological membrane is calculated by comparing with a blank group.
Thioflavin T (ThT) is selected as a bacterial membrane potential indicator to investigate the change of the bacterial membrane potential after the treatment of the silver nanoenzyme assembly. ThT is a positively charged small molecule dye that can enter bacteria by diffusion. When ribosome dysfunction causes a decrease in bacterial membrane protein synthesis and an increase in bacterial membrane potential, thT accumulates in the bacteria to a greater extent, and a strong fluorescence signal is observed when the bacteria are observed. Thioflavin T (ThT) is added into the three groups of culture solutions simultaneously to be used as a bacterial membrane potential indicator, and the final concentration of ThT is 10 mu M. And (3) incubating for 12h at 37 ℃, taking the treated bacterial liquid every other hour, and measuring the fluorescence value of ThT and the OD600 of the bacterial liquid. When a spectrofluorometer detects ThT fluorescence, the excitation wavelength is set to 440nm and the emission wavelength is set to 494nm.
Incubating the above three groups of culture solutions at 37 deg.C for 48h, centrifuging, washing for 3 times, adding extracellular matrix dye canavalin A-rhodamine isothiocyanate, incubating and dyeing at room temperature for 25min, centrifuging (5000rpm, 5min,4 deg.C), collecting the formed biological membrane in the container, resuspending with PBS, measuring fluorescence intensity (excitation wavelength 550nm, emission wavelength 580 nm) by fluorescence spectrophotometer, and measuring OD 600 The total amount of bacteria was quantified and the mean fluorescence intensity was obtained as the ratio of fluorescence intensity to OD600. The level of extracellular matrix secretion was evaluated by calculating the relative fluorescence intensity of each of the other groups with the average fluorescence intensity of the blank control group being 1.
The three groups of culture solutions are incubated at 37 ℃ for 48h, centrifuged and washed for 3 times, then added with a beta-lactamase indicator nitrothiophene for incubation for 3h, and the absorbance of the beta-lactamase indicator nitrothiophene at 490nm is measured by an ultraviolet spectrophotometer. Simultaneously by measuring OD 600 The total amount of bacteria was quantified and the mean fluorescence intensity was obtained by the ratio of fluorescence intensity to OD600. Relative absorbance of the other groups was calculated with the average absorbance of the blank control group being 1, to thereby evaluate the relative activity percentage of beta-lactamase.
The results are shown in fig. 6, the silver nanoenzyme assembly can generate ROS in an environment simulating a drug-resistant bacterial biofilm; the silver nanoenzyme assemblies can reduce the total amount of bacterial biofilm, and this effect can be inhibited by ROS scavengers (catalase).
The results are shown in fig. 7, the silver nanoenzyme assembly can cause bacterial membrane hyperpolarization and inhibit production of extracellular matrix and beta-lactamase by drug-resistant bacteria.
Example 3: the effect of the silver nanoenzyme assembly/antibiotic combination drug in the treatment of drug-resistant bacterial biofilm infection.
Preparation of the medicament: the silver nanoenzyme assembly prepared in example 1 was diluted in TSB to a final concentration of 40 μ g/mL, and ampicillin was weighed and dissolved in TSB to a final concentration of 5 μ g/mL, to obtain a silver nanoenzyme assembly/antibiotic combination.
Establishing a drug-resistant bacterial biofilm model: taking a proper amount of methicillin-resistant staphylococcus aureus suspension, and diluting the suspension by using a TSB culture medium until the concentration of the bacterial liquid is about 2 multiplied by 10 5 CFU/mL, inoculating into a glass bottom culture dish, and culturing in a constant temperature incubator at 37 ℃ for 48h to form a mature bacterial biofilm.
Group setting:
a. control group: the mature biofilms were given fresh TSB.
b. Treatment group 1: ampicillin-containing TSB (ampicillin final concentration of 5. Mu.g/mL) was added to the mature biofilms.
c. Treatment group 2: adding Ag into mature biological membrane + TSB (final silver concentration 40. Mu.g/mL).
d. Treatment group 3: TSB containing silver nanoenzyme assemblies (final silver concentration 40. Mu.g/mL) was added to the mature biofilms.
e. Treatment group 4: adding ampicillin and Ag into mature biological membrane + The TSB of (1) (final concentration of ampicillin was 5. Mu.g/mL, silver concentration was 40. Mu.g/mL).
f. Treatment group 5: adding the TSB of the silver nano enzyme assembly/antibiotic combined drug into the mature biomembrane.
After the mature bacterial biofilm was incubated with different treatment groups of antibacterial agents for 24h, the well plate was inverted and the supernatant was discarded, and washed 3 times with physiological saline. Subsequently, a physiological saline solution containing Syto Green (4.5. Mu.M) and PI (0.05 mg/mL) was added and incubated at 37 ℃ for 20min. After the dyeing is finished, washing off redundant dye, and sealing the piece by using an anti-fluorescence quenching sealing piece agent. Two kinds of fluorescence in the biological membrane are observed and photographed through a laser confocal microscope.
The results are shown in fig. 8, the silver nanoenzyme assembly/antibiotic combination drug can effectively kill drug-resistant bacteria in mature biofilms.
Example 4: drug resistance reversal effect of silver nano enzyme assembly/antibiotic combined drug in mouse skin drug-resistant bacterial biofilm infection
Preparation of the medicament: dispersing the silver nanoenzyme assembly prepared in example 1 into a phosphate buffer solution, weighing ampicillin and dispersing the ampicillin into the phosphate buffer solution, and mixing the two solutions according to the ratio of 8:1 mass ratio to obtain the silver nano enzyme assembly/antibiotic combined drug.
Establishing a skin infection animal model: taking 6-8 week old balb/c female mice as model animals, and adjusting the concentration of methicillin-resistant staphylococcus aureus suspension to 10 bacterial liquid concentration 8 CFU/mL, was injected subcutaneously (100. Mu.L/mouse) in the right dorsal side of the mice. After 24h, the injection site was observed to appear white in appearance, indicating successful molding.
Group setting:
a. control group: after the successful establishment of the model is confirmed, physiological saline is injected subcutaneously at the skin infection part of the mouse.
b. Treatment group 1: after the model was successfully established, a liquid containing ampicillin (ampicillin concentration: 0.27 mg/kg) was injected subcutaneously into the skin infection site of the mouse.
c. Treatment group 2: after the model is successfully established, ag is injected into the skin infection part of the mouse subcutaneously + The chemical solution (silver concentration: 2.16 mg/kg) of (1).
d. Treatment group 3: after the model is successfully established, the liquor containing the silver nano enzyme assembly is injected into the infected part of the mouse skin subcutaneously (the silver concentration is 2.16 mg/kg).
e. Treatment group 4: after the model is successfully established, ampicillin and Ag are injected into the skin infection part of the mouse subcutaneously + The liquid medicine (ampicillin concentration: 0.27mg/kg, silver concentration: 2.16 mg/kg).
f. Treatment group 5: after the model is successfully established, the liquid medicine containing the silver nano enzyme assembly/antibiotic combined medicine is injected into the infected part of the mouse skin subcutaneously (the concentration of ampicillin is 0.27mg/kg, and the concentration of silver is 2.16 mg/kg).
The area of the skin infected by the mice is counted during the treatment process. On day 15 of treatment, mice were sacrificed, infected skin was collected, weighed, homogenized, and the homogenate was diluted by multiple (1-fold, 10-fold, 102-fold, 103-fold, respectively) and spread on TSB solid medium, which was then placed at 37 ℃ for 24h of inverted incubation. Finally, the colony formation on the solid culture medium is counted to determine the number of bacteria in the skin so as to investigate the effect of the silver-silver nanoenzyme assembly/antibiotic combined drug on treating skin infection.
The results are shown in fig. 9, where the silver nanoenzyme assembly/antibiotic combination drug was able to synergistically treat drug-resistant bacterial biofilm infections.
The invention proves the activity of the analogous oxidase and the activity of the analogous thiol oxidase of the silver nano enzyme assembly through two aspects of material characterization experiments and in-vivo and in-vitro experiments, and verifies the effects of the silver nano enzyme assembly in the silver nano enzyme assembly/antibiotic combined medicine on destroying bacterial biofilms and inhibiting bacterial secretion functions, and finally verifies the effect of the silver nano enzyme assembly/antibiotic combined medicine on treating drug-resistant bacterial biofilm infection in-vitro and in-vivo, thereby providing a potential nano material for the research of the antibiotic combined medicine for clinically resisting the drug-resistant bacterial infection.
Claims (7)
1. The application of the silver nanoenzyme assembly/antibiotic in preparing the combined medicament for treating the drug-resistant infectious diseases is characterized in that the combined medicament comprises the silver nanoenzyme assembly and the antibiotic, and the silver nanoenzyme assembly is composed of ultra-small silver nanoenzyme, infection microenvironment responsive macromolecular ligand and amphiphilic surfactant.
2. The use of the silver nanoenzyme assembly/antibiotic of claim 1 in the preparation of a combination medicament for the treatment of drug-resistant infectious diseases, wherein the preparation method of the silver nanoenzyme assembly comprises:
1) Preparing the ultra-small silver nano enzyme: preparing ultra-small silver nanoenzyme by a thermal decomposition method by taking silver nitrate as a precursor, oleylamine as a reducing agent and oleic acid as a solvent;
2) Preparing a silver nano enzyme assembly: mixing the ultra-small silver nanoenzyme and the infection microenvironment responsive macromolecular ligand in an organic solvent, slowly dropping the mixture into phosphate buffer solution dissolved with amphiphilic surfactant in water bath ultrasound, fully emulsifying, and volatilizing to remove the organic solvent to obtain the silver nanoenzyme assembly.
3. The use of the silver nanoenzyme assembly/antibiotic of claim 1 in the preparation of a combination drug for the treatment of drug-resistant infectious diseases, wherein in step 1), the mass ratio of silver nitrate to oleylamine is 1:2.9-5.9, and the heating rate is 1-10 ℃/min.
4. Use of the silver nanoenzyme assembly/antibiotic of claim 1 in the preparation of a combination drug for the treatment of drug-resistant infectious diseases, wherein in step 2) the responsive group in the infection microenvironment-responsive polymeric ligand comprises one or more of gluconic acid, imidazole, hyaluronic acid, poly (epsilon-caprolactone), polyphosphate, gelatinase-cleavable peptide;
the amphiphilic surfactant is selected from one or more of poloxamer, polyethylene glycol-polystyrene and polyethylene glycol-polylactic acid.
5. The application of the silver nanoenzyme assembly/antibiotic in preparing a combined medicament for treating drug-resistant infectious diseases according to claim 1, wherein the size of the ultra-small silver nanoenzyme is 3-5nm, the hydrated particle size of the silver nanoenzyme assembly obtained by assembling an infection microenvironment responsive polymeric ligand and an amphiphilic surfactant is 200nm, and the hydrated particle size after the disassembly is 10-20nm.
6. The use of the silver nanoenzyme assembly/antibiotic of claim 1 in the preparation of a combination medicament for the treatment of drug-resistant infectious disease, wherein the antibiotic is ampicillin, penicillin, streptomycin.
7. The use of the silver nanoenzyme assembly/antibiotic of claim 1 in the preparation of a combination for the treatment of drug-resistant infectious diseases, wherein the mass ratio of the silver nanoenzyme assembly to the antibiotic in the combination is 8.
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