Antibiotic nano-enzyme capable of reversing drug resistance

Abstract

The invention discloses an antibiotic nano-enzyme capable of reversing drug resistance, and relates to the technical field of biological medicines, and the antibiotic nano-enzyme is formed by assembling an antibiotic and hemin. The antibiotic and hemin are assembled to form the nano-enzyme to reverse the drug resistance of the antibiotic, and the prepared antibiotic nano-enzyme has an excellent antibacterial effect.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly relates to an antibiotic nanozyme for reversing drug resistance and a preparation method thereof. Background Art

[0002] Antibiotic resistance poses a major threat to public health and the environment, and the rapid emergence of multi-drug resistant bacteria is endangering the effectiveness of antibiotic treatment. One of the main mechanisms of antibiotic resistance is the reduced permeability of the bacterial cell membrane, resulting in decreased drug uptake. Currently, the strategies for the modification and development of antibiotics mainly include β-lactamase inhibitors, siderophore-conjugated antibiotics, antibody-antibiotic conjugates, proton pump inhibitor combinations, and immune activator combinations, etc. However, the narrow spectrum of their application and the single antibacterial mechanism still make it difficult to avoid the recurrence of drug resistance.

[0003] Nanozymes are a new generation of artificial enzymes with unique physical and chemical properties and enzyme-like catalytic activities. Nanozymes with peroxidase (POD) or oxidase (OXD) activities can generate a large amount of reactive oxygen species (ROS) to combat bacteria. In addition, nanozymes with deoxyribonuclease (DNase)-like activities can also prevent the spread of drug resistance by degrading bacterial drug resistance genes. Compared with natural enzymes, nanozymes are generally more stable, economical, and practical. Usually, compared with natural enzymes, nanozymes are more stable, more economical, and more practical. Therefore, nanozyme-based antibacterial alternatives (Nanozybiotics) may have greater application potential in the antibacterial field.

[0004] Heme is an important substance in the human body, serving as a cofactor for the catalytic action of various enzymes and playing a role in the transport of gas molecules. The catalytic mechanism of heme mainly depends on the regulation of its axial coordination by histidine, while the catalytic ability of the planar Fe-N4 structure of heme is limited. In addition, heme can act as a siderophore to transport antibiotics. Therefore, we propose a scheme for designing a new type of antibiotic nanozyme, that is, assembling various antibiotics with heme through a coordination-precipitation method to improve the uptake of antibiotics by bacteria and solve the problems existing in current research strategies. Summary of the Invention

[0005] In order to solve the above technical problems, the object of the present invention is to provide an antibiotic nanozyme for reversing drug resistance. The present invention reverses the drug resistance of antibiotics by assembling antibiotics with hemin to form a nanozyme, and the prepared antibiotic nanozyme has excellent antibacterial effects.

[0006] The technical solution for the present invention to solve the above technical problems is as follows: providing an antibiotic nanozyme for reversing drug resistance, which is assembled from an antibiotic and hemin.

[0007] Further, the antibiotic is at least one of fluconazole, vancomycin, ceftriaxone sodium, ampicillin, azithromycin, novobiocin, cephalexin, and gentamicin.

[0008] Further, the preparation method of the antibiotic nanozyme comprises the following steps:

[0009] (1) Dissolve hemin in dimethyl sulfoxide to obtain a hemin solution;

[0010] (2) Dissolve the antibiotic in ultrapure water to obtain an antibiotic solution;

[0011] (3) Mix the hemin solution obtained in step (1) with the antibiotic solution obtained in step (2), then adjust the pH to 6.5 - 7.5 with sodium hydroxide solution, seal and stir overnight at room temperature, and obtain the antibiotic nanozyme with reversed drug resistance through centrifugation and vacuum freeze-drying.

[0012] Furthermore, in step (3) of the preparation method, the concentration of the hemin solution is 0.04 - 0.06 mM / mL. Preferably, the concentration of the hemin solution is 0.05 mM / mL.

[0013] Furthermore, in step (3) of the preparation method, the concentration of the antibiotic solution is 0.08 - 0.12 mM / mL. Preferably, the concentration of the antibiotic solution is 0.1 mM / mL

[0014] Furthermore, in step (3) of the preparation method, the volume ratio of the hemin solution to the antibiotic solution is 1:0.8 - 1.2. Preferably, the volume ratio of the hemin solution to the antibiotic solution is 1:1.

[0015] Furthermore, in step (3) of the preparation method, the concentration of the sodium hydroxide solution is 0.8 - 1.2 mol / L.

[0016] The present invention has the following beneficial effects:

[0017] 1. The antibiotic nanozyme designed by the present invention retains the antibiotic component, can replace histidine through axial coordination, and thus has catalytic properties. Therefore, it can degrade the biofilm and change the permeability of the bacterial membrane to enhance the absorption of antibiotics.

[0018] 2. Due to the functional groups of hemin and its ability to coordinate with gas molecules, it can bind to cystathionine-γ-lyase (CSE) and H 2 S in bacteria, thereby regulating the sensitivity of bacteria to antibiotics at the molecular level and further enhancing the absorption of antibiotics.

[0019] 3. The interaction between the antibiotic nanozyme of the present invention and fungi can induce ferroptosis in fungi, and its biological characteristics include elevated ROS, lipid peroxidation, cell membrane shrinkage, GSH consumption, and gene levels, etc.

[0020] 4. The synthesis method of the antibiotic nanozyme based on molecular self-assembly is easy to obtain or mass-produce. The antibiotic nanozyme can be applicable to various antibiotics, and different additives can be selected to expand the scope of the antibiotic nanozyme, thus having a certain universality. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a molecular model of levofloxacin hydrochloride nanozyme and a detection result graph of POD activity;

[0022] Figure 2 It is a molecular model of fluconazole nanozyme and a detection result graph of POD activity;

[0023] Figure 3 It is a molecular model of hydroxypyridine thione nanozyme and a detection result graph of POD activity;

[0024] Figure 4 It is a molecular model of rapamycin nanozyme and a detection result graph of POD activity;

[0025] Figure 5 It is a molecular model of gentamicin nanozyme and a detection result graph of POD activity;

[0026] Figure 6 It is a heat map of the affinity ranking after the assembly of different antibiotics and heme;

[0027] Figure 7 It is a graph of the POD activity and bactericidal effect of benzathine penicillin nanozyme;

[0028] Figure 8 It is a graph of the POD activity and bactericidal effect of polymyxin B nanozyme;

[0029] Figure 9 It is a graph of the elemental analysis result of gentamicin nanozyme;

[0030] Figure 10 It is an ultraviolet full-wavelength absorption spectrum graph before and after the assembly of gentamicin and hemin chloride;

[0031] Figure 11 It is a Fourier transform infrared spectrum graph before and after the assembly of gentamicin and hemin chloride;

[0032] Figure 12 It is a transmission electron microscope observation graph of the morphological structure of MRSA treated with gentamicin nanozyme;

[0033] Figure 13Quantitative detection result graph of protein leakage of MRSA;

[0034] Figure 14 Quantitative result graph of ROS fluorescence intensity in the antibacterial experiment of gentamicin nanozyme;

[0035] Figure 15 Colony count statistical graph of the antibacterial experiment of gentamicin nanozyme combined with H2O2;

[0036] Figure 16 MRSA membrane potential change graph in the antibacterial experiment of gentamicin nanozyme;

[0037] Figure 17 For the fluorescent probe BODIPY TM Result graph of the lipid peroxidation level in bacteria after treating MRSA with gentamicin nanozyme detected by 581 / 591C11;

[0038] Figure 18 MDA detection result graph in the antibacterial experiment of gentamicin nanozyme;

[0039] Figure 19 GSH-Px enzyme activity detection result graph in the antibacterial experiment of gentamicin nanozyme;

[0040] Figure 20 For the content detection result graph of H 2 S in the antibacterial experiment of gentamicin nanozyme;

[0041] Figure 21 Using AI to screen drugs approved by the FDA targeting Candida albicans glucan;

[0042] Figure 22 Molecular structure schematic diagrams of rapamycin and ceftriaxone sodium;

[0043] Figure 23 Antibacterial effect test result graph of antibiotic nanozyme against Candida albicans;

[0044] Figure 24 Test result graph of antibiotic nanozyme against methicillin-resistant Staphylococcus aureus;

[0045] Figure 25 Test result graph of antibiotic nanozyme against drug-resistant Escherichia coli;

[0046] Figure 26 Peroxidase activity detection result graph of antibiotic nanozyme;

[0047] Figure 27 Result graph of detecting the total ROS level in Candida albicans using a fluorescence microscope

[0048] Figure 28It is the ROS fluorescence quantitative result diagram of the antibacterial experiment of antibiotic nanozymes;

[0049] Figure 29 It is the result diagram of the lipid peroxidation level in fungi after treating Candida albicans with fluconazole nanozyme and ceftriaxone sodium nanozyme detected by the fluorescent probe BODIPY TM 581 / 591C11;

[0050] Figure 30 It is the result diagram of MDA detection in the antibacterial experiment of antibiotic nanozymes;

[0051] Figure 31 It is the result diagram of the determination of the GSH level in fungi;

[0052] Figure 32 It is the SEM observation diagram after treating Candida albicans with fluconazole nanozyme;

[0053] Figure 33 It is the antibacterial result diagram after treating Candida albicans with ferroptosis inducer combined with fluconazole or ceftriaxone sodium;

[0054] Figure 34 It is the Volcano diagram of all differentially expressed genes in Candida albicans treated with fluconazole nanozyme compared with fluconazole treatment;

[0055] Figure 35 It is the result diagram of GO annotation analysis of differentially expressed genes in Candida albicans treated with fluconazole nanozyme compared with fluconazole treatment;

[0056] Figure 36 It is the KEGG enrichment diagram of differentially expressed genes in Candida albicans treated with fluconazole nanozyme compared with fluconazole treatment;

[0057] Figure 37 It is the result diagram of the differential expression analysis of ferroptosis-related genes after treating Candida albicans with fluconazole nanozyme;

[0058] Figure 38 It is the heat map of the differential expression of genes encoding efflux pumps in fungi after treating Candida albicans with fluconazole nanozyme;

[0059] Figure 39 It is the result diagram of the detection of L-LDH level in serum after treatment with fluconazole nanozyme;

[0060] Figure 40 It is the 14-day body weight development change diagram of mice after intranasal inoculation with low and high doses of fluconazole nanozyme and ceftriaxone sodium nanozyme;

[0061] Figure 41 It is the result diagram of blood routine and biochemical analysis on the 15th day after treating normal healthy mice by intranasal dripping with different drugs;

[0062] Figure 42 Representative H&E staining images of the heart, liver, spleen, lungs, and kidneys of normal healthy mice on the 15th day after nasal instillation of different drugs.

[0063] Figure 43 Schematic diagram of the experiment to evaluate the therapeutic effect of fluconazole nanozyme on wound infection in mice caused by Candida albicans.

[0064] Figure 44 Representative images of the infected wounds of mice at different times during the treatment period.

[0065] Figure 45 Graph showing the relative area percentage and healing rate of the infected wounds of mice at different times during the treatment period.

[0066] Figure 46 Graph showing the detection results of fungal load in the wounds of mice with wound models after the treatment.

[0067] Figure 47 Graph showing the detection results of fungal load in the lung tissues of mice with pneumonia models after the treatment.

[0068] Figure 48 Representative H&E staining images of the heart, liver, spleen, lungs, and kidneys of different drug treatment groups after the treatment of pneumonia mice.

[0069] Figure 49 Graph showing the detection results of fungal load in the vaginal lavage fluid of mice with vaginitis models after the treatment. Detailed implementation methods

[0070] The principles and characteristics of the present invention are described below. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention. For those not specified in the examples, the conventional conditions or the conditions recommended by the manufacturer are followed. For the reagents or instruments not specified for the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0071] Example 1

[0072] Antibiotic nanozymes with reversed drug resistance were assembled using antibiotics such as fluconazole, ceftriaxone sodium, and gentamicin, and hemin.

[0073] The preparation method includes the following steps:

[0074] (1) Dissolve hemin in dimethyl sulfoxide to obtain a hemin solution (0.05 mM / mL).

[0075] (2) Dissolve the antibiotic in ultrapure water to obtain an antibiotic solution (0.1 mM / mL).

[0076] (3) Mix the hemin solution obtained in step (1) and the antibiotic solution obtained in step (2) at a volume ratio of 1:1, then adjust the pH to 7 with sodium hydroxide solution (1M), seal it, stir overnight at room temperature, and obtain the antibiotic nanozyme with reversed drug resistance after centrifugation and vacuum freeze-drying.

[0077] Experimental Example 1 Hemin and various antibiotic molecules can co-assemble to form nanozymes with catalytic ability

[0078] (1) We first detected the POD activities of quinolone, azole, pyrithione, hydroxyl, and amino drugs after assembly with hemin, and the results are as Figure 1-5 shown. Then, taking various types of antibiotics such as aminoglycoside antibiotic gentamicin, β-lactam antibiotic ampicillin, glycopeptide antibiotic vancomycin, and cationic polypeptide polymyxin B as examples, by detecting the affinity between the antibiotic monomers and hemin, the enzyme activity level after the assembly of the antibiotic and hemin was predicted, and the results are as Figure 6 shown. It was found that benzathine penicillin had the highest affinity with hemin, and polymyxin B had the lowest affinity with hemin. Then we detected the POD activities and bactericidal effects of benzathine penicillin and polymyxin B after assembly with hemin, and the results are as Figure 7 and Figure 8 shown, and it was found that the results were consistent with the affinity prediction described above. In addition, we observed that polymyxin B could not form a Soret band with hemin chloride, indicating that it could not form an axial coordination and catalytic active center. Therefore, polymyxin B (hemin) did not enhance its bactericidal ability.

[0079] Experimental Example 2 Take gentamicin nanozyme as an example to characterize the antibiotic nanozyme.

[0080] The results showed the cross-linked spherical structure of gentamicin nanozyme, with a scale in the nanometer level. High-angle annular dark-field images observed uniformly arranged Fe atoms in the structure of gentamicin nanozyme at a scale of 5 nm. As Figure 9 shown, in the elemental analysis results, Fe, C, N, and O elements were uniformly distributed in the structure of gentamicin nanozyme, and the results showed that the Fe element in hemin chloride was uniformly distributed in gentamicin nanozyme, proving the uniform assembly of hemin chloride and gentamicin.

[0081] (2) By detecting the ultraviolet full-wavelength absorption spectra of gentamicin before and after assembly with hemin chloride, analyze the characteristic absorption peaks of the two to judge the structural changes that occurred before and after assembly. As Figure 10As shown, the results indicate that before assembly, gentamicin has a characteristic absorption peak at 240 nm due to its aminoglycoside structure, while hemin has several weak absorptions in the range of 500 - 750 nm due to its porphyrin ring structure, known as Q bands. After assembly, the characteristic peak of gentamicin undergoes a red shift to approximately 260 nm, suggesting a change in its structure. After assembly, hemin shows a Soret band at 415 nm, and the absorption of the Q bands decreases, indicating that the Fe atom at the center of the porphyrin ring of hemin coordinates, and the planar porphyrin structure undergoes an axial shift. This result proves that hemin coordinates with gentamicin through the Fe atom at its porphyrin center.

[0082] In addition, to further explore the structural changes, a Fourier transform infrared spectrometer was used to detect the gentamicin nanozyme before and after assembly, and the results are shown as Figure 11 shown. Characteristic absorption peaks of gentamicin and hemin appear at 2000 - 2500 cm -1 and 2500 - 3000 cm -1 respectively, which means the formation of a co-assembled system. The disappearance of the peak of N-H bending at 1634 cm -1 indicates that Fe on hemin and NH of gentamicin 2 may form a coordination bond, which further affects the stretching of the adjacent functional group C-O of gentamicin. The disappearance of the C=O stretching of hemin and the peak of O-H at 3433 cm -1 indicates that intermolecular hydrogen bonds may be formed at the carboxyl ends at both ends of hemin, thus forming a 3D nanostructure.

[0083] (3) Taking the gentamicin nanozyme as an example, to visually observe the morphological and structural changes of MRSA before and after treatment with the gentamicin nanozyme, after treatment, the MRSA bacteria were fixed. After dehydration, embedding, and ultramicrotomy or dehydration and critical point drying, a scanning electron microscope was used to observe the morphology of MRSA bacteria, and the results are as Figure 12 shown. The shapes of the bacteria in the blank control, gentamicin control, and hemin control are plump, the cell walls and cell membranes are intact, and the cytoplasm is relatively uniform, while the shapes of the bacteria in the gentamicin nanozyme treatment group are shrunk, the cell membranes and walls are ruptured, the cytoplasm has uneven light and dark, and the bacterial contents leak out.

[0084] (4) To further verify the bactericidal effect of the gentamicin nanozyme on bacteria, a protein quantification test was performed on the treated bacterial suspension to quantitatively verify the results of the destruction of bacterial cells and the leakage of contents, and the results are as Figure 13As shown, the protein content in the MRSA system treated with gentamicin nanozyme was significantly higher than that in the control group, indicating that the MRSA cells were damaged and cytoplasmic proteins leaked into the system. In contrast, there was no significant difference in protein quantification between the gentamicin control group and the heme control group compared with the blank control group, suggesting that cytoplasmic protein leakage was not obvious and the bacterial cells were not ruptured.

[0085] (5) To further verify the ROS generated by gentamicin nanozyme during the anti-MRSA effect, the ROS of bacteria treated with gentamicin nanozyme was detected by fluorescence using the probe DCFH-DA. First, the overnight cultured bacterial solution was transferred and activated for 4 h (about 1×10 9 CFU mL -1 ), centrifuged at 5000 rpm for 3 min in a centrifuge, and the obtained bacterial pellet was resuspended with an equal volume of ddH 2 O. In 1.5 mL sterile centrifuge tubes, 900 μL of bacterial suspension and 100 μL of gentamicin nanozyme solution or single-component solution (1 mg / mL) were added respectively, mixed well, and incubated in a 37°C constant temperature incubator for 3 h. Then, the DCFH-DA dye was added to each centrifuge tube, incubated in the dark at 37°C for 30 min, centrifuged at 5000 rpm for 3 min, and resuspended with PBS. The washing was repeated 3 times. The precipitate of each group was resuspended with 100 μL of PBS, 20 μL was pipetted and dropped on a clean glass slide, 5 μL of anti-fluorescence quenching mounting solution was added and mixed well, and then a cover glass was covered. Finally, imaging was performed using a laser confocal microscope. The results are as Figure 14 shown. More ROS fluorescence appeared in the MRSA bacterial cells treated with gentamicin nanozyme, while this phenomenon did not occur in the three control groups, proving that the peroxidase activity of gentamicin nanozyme can significantly increase the generation of bacterial ROS and plays a key role in the oxidative damage of bacteria, serving as one of the mechanisms for reversing MRSA drug resistance and effective antibacterial action.

[0086] In the in vitro antibacterial experiment, it was also explored whether the peroxidase activity of gentamicin nanozyme played a role in killing MRSA. Therefore, a lower concentration of H 2 O 2 (50 μM) was added, and the antibacterial experiment was carried out and counted by plate coating. The results are as Figure 15 shown. After adding the additional H 2 O 2 , the antibacterial effect of gentamicin nanozyme on MRSA was significantly improved, and the bacterial activity decreased by 10 times. This proved that the peroxidase activity of gentamicin nanozyme participated in the role of reversing MRSA drug resistance, catalyzed H 2 O 2 to generate ROS, and exacerbated the killing effect on bacteria.

[0087] (6)In the oxidative damage of bacteria, the change in membrane potential is an observation index for observing the damage of bacterial biofilms. As a cationic probe sensitive to membrane potential, DISC3(5) shows enhanced fluorescence signal when the membrane potential is lost. Therefore, the change in membrane potential is determined by the change in the fluorescence intensity of DISC3(5). The results are as Figure 16 shown. The depolarization of the membrane potential of bacteria treated with gentamicin nanozyme was significant, while no obvious changes were observed in the three control groups, proving that the action of gentamicin nanozyme led to the loss of bacterial membrane potential, suggesting the occurrence of membrane damage.

[0088] To further confirm the occurrence of oxidative damage to bacterial biofilms, the lipid peroxidation probe BODIPY TM 581 / 591C11 was used to detect the content of peroxidized lipids. The results of membrane lipid peroxidation detected by BODIPY are as Figure 17 shown. In MRSA treated with gentamicin nanozyme, the content of peroxidized lipids increased significantly, proving the occurrence of bacterial membrane peroxidation during the anti-MRSA action of gentamicin nanozyme. In addition, an increase in peroxidized lipids was also observed in the bacteria in the gentamicin treatment group, which may be related to the effect of gentamicin on damaging the cell membrane.

[0089] In addition, the increase in bacterial lipid peroxidation after treatment with gentamicin nanozyme was also detected by directly detecting the lipid peroxidation product malondialdehyde (MDA). The results are as Figure 18 shown. The content of MDA in the MRSA cells in the gentamicin nanozyme treatment group increased significantly, confirming the occurrence of bacterial lipid peroxidation and also proving that the peroxidase activity of gentamicin nanozyme can cause lipid peroxidation in the cells, which is an important mechanism involved in antibacterial action.

[0090] (7)Reduced glutathione (GSH) is a small mercapto molecule widely present in plants, animals, fungi, and bacteria. As an antioxidant, it resists the action of reactive oxygen species. In bacteria, GSH can also activate the expression of virulence genes and promote the formation of biofilms. Due to the peroxidase activity of gentamicin nanozyme, we detected its peroxidation effect on GSH substrates to explore the possibility of promoting antibacterial action by reducing the bacterial GSH level. The results are as Figure 19As shown, when using GSH as a substrate, the gentamicin nanozyme exhibits peroxidase activity and can significantly scavenge GSH. This indicates that in antibacterial action, the gentamicin nanozyme also exerts GSH-Px enzyme activity, which is another antibacterial mechanism of the enzyme catalytic activity of the gentamicin nanozyme besides ROS regulation. The results show that after introducing exogenous GSH, the antibacterial effect of the gentamicin nanozyme is reversed. This result proves that GSH not only directly detoxifies the action of ROS as an antioxidant but also competes with the gentamicin nanozyme as a substrate, weakening its antibacterial effect.

[0091] (8) Besides GSH, hydrogen sulfide (H 2 S) is also an important type of small molecule thiol in bacterial drug resistance. When bacteria encounter stimuli and damage, they will produce more H 2 S gas molecules to counteract oxidative stress and acquire tolerance. Therefore, the fluorescence probe WSP-5 was used to detect the H 2 S content in MRSA bacteria after treatment with the gentamicin nanozyme, and the results are as Figure 20 shown. The results show that the H 2 S level in bacteria significantly decreases after treatment with the gentamicin nanozyme, proving that the gentamicin nanozyme can inhibit the production of bacterial H2S or consume H 2 S, thereby reversing bacterial drug resistance and enhancing the antibacterial effect.

[0092] Furthermore, through experiments, it was also verified that the gentamicin nanozyme may inhibit the enzyme activity of CSE by binding to pyridoxal phosphate (PLP), thereby reducing the H 2 S level.

[0093] Experimental Example 3

[0094] To evaluate the in vitro antifungal ability of the antibiotic nanozyme, as Figure 21 shown, we used AI to screen out rapamycin and ceftriaxone sodium, drugs approved by the FDA that target glucan in Candida albicans (as Figure 22 shown). Three drug-resistant Candida albicans strains, drug-resistant C.albicans ATCC 90029, 90028, and 10231, were used as model pathogens for verification. The growth curves of drug-resistant Candida albicans under different treatments were observed using different concentrations of the antibiotic nanozyme and the same concentration of the antibiotic solution. The specific steps are as follows: Three single colonies of drug-resistant Candida albicans ATCC 90029, 90028, and 10231 were respectively inoculated into SDB liquid medium and cultured overnight in a constant temperature shaker at 37°C and 220 rpm. The next day, they were diluted to fresh SDB liquid medium at a volume ratio of 1:100 and activated in a constant temperature shaker at 37°C and 220 rpm for 6 h (about 1×10 8 CFU mL-1 )Spare

[0095] Set the concentration gradient to 100 μg / mL -1 , 50 μg / mL -1 , 10 μg / mL -1 , 5 μg / mL -1 and 1 μg / mL -1 of ceftriaxone sodium nanozyme CRO (Hemin), and set the same series of concentrations of ceftriaxone CRO, Hemin, and the corresponding resistant antibacterial agents (itraconazole, 5-fluorocytosine, fluconazole) of the three strains as negative controls, and dissolve them with SDB liquid medium respectively. The antibacterial reaction system was carried out in a 15 mL sterile shaking tube. Add 100 μL of bacterial liquid, 100 μL of antibiotic nanozyme or other components, and 800 μL of SDB liquid medium into the tube. 100 μL of bacterial liquid plus 900 μL of SDB liquid medium was used as a blank control. Each centrifuge tube was placed in a constant temperature shaker at 37 °C and 220 rpm. At the 24th hour, 200 μL of the suspension was taken and placed in a 96-well plate respectively. A multifunctional microplate reader was used to detect the absorbance at 600 nm to determine the minimum concentration that inhibits fungal growth after 24 h at 37 °C. The determination was carried out three times in the 96-well plate. Next, set the concentration gradient to 1 μg / mL -1 , 0.5 μg / mL -1 , 0.1 μg / mL -1 , 0.05 μg / mL -1 and 0.01 μg / mL -1 of rapamycin nanozyme RAPA (Hemin), and the test method was the same as that of CRO (Hemin). The results are as Figure 23 shown. Antibiotics and hemin at the same concentration did not show better antifungal effects than antibiotic nanozymes. At the 24th hour, the inhibitory effects of CRO (Hemin) and RAPA (Hemin) on the growth of these three drug-resistant Candida albicans were better than those of other control groups at the same concentration, verifying that antibiotic nanozymes have excellent antibacterial activity and reversing the drug resistance of drug-resistant Candida albicans.

[0096] Experimental Example 5

[0097] To verify whether antibiotic nanozymes have broad-spectrum antibacterial activity, the antibacterial activities of four antibiotic nanozymes, namely ceftriaxone nanozyme (Hemin), ampicillin nanozyme AMP (Hemin), gentamicin nanozyme GM (Hemin), and vancomycin nanozyme VAN (Hemin), were also detected using methicillin-resistant Staphylococcus aureus and drug-resistant Escherichia coli. In this study, the blank control group, Hemin group, antibiotic monomer group, and antibiotic nanozyme group were detected. The test results of methicillin-resistant Staphylococcus aureus and drug-resistant Escherichia coli are as Figure 24 andFigure 25 shown.

[0098] Depend on Figure 24-25 It can be seen that compared with other control groups, the growth of MRSA and drug-resistant E. coli in the logarithmic growth phase was significantly inhibited when co-treated with 500μg / mL antibiotic nanozymes. This result also proves that antibiotic nanozymes have a reversal effect on MRSA and drug-resistant E. coli during the growth period of the culture medium. The results show that antibiotic nanozymes have strong broad-spectrum antibacterial activity.

[0099] Test Example 6

[0100] In order to explore the mechanism of action of antibiotic nanozymes against drug-resistant bacteria, verification was carried out with reference to Experimental Example 2.

[0101] (1) Figure 26 As shown, using TMB as a colorimetric assay, it was verified that the antibiotic nanozymes GM (Hemin), 5-FC (Hemin), CA (Hemin), Lawsone (Hemin) and Pyrithione (Hemin) all have peroxidase activity. The ROS produced by POD activity can directly increase the oxidative damage of fungi and directly kill the fungi; in addition, this activity can catalyze membrane lipid peroxidation to cause damage to the fungal cell membrane and increase permeability, thereby increasing the fungal uptake of antibiotics.

[0102] (2) To test whether the catalytic activity of antibiotic nanozymes can promote the production of ROS and thus inhibit the growth of Candida albicans, the probe 2',7'-dichlorodihydrofluorescein DCFH-DA was used to detect the reactive oxygen species (ROS) in fungi. DCFH-DA can be converted into 2',7' dichlorofluorescein (DCF) after reacting with ROS. Figure 27 and Figure 28 As shown, fluorescence microscopy imaging clearly shows that 1 mg / mL fluconazole nanozyme and fungus treatment produces more ROS fluorescence compared to other treatments, indicating that it has the strongest ability to produce intracellular ROS in Candida albicans. It proves that the peroxidase activity of fluconazole nanozyme can significantly increase the generation of fungal ROS, playing a key role in the oxidative damage of fungi, as one of the mechanisms for reversing drug resistance and effectively fighting against Candida albicans.

[0103] (3) Lipid peroxide accumulation is the core mechanism of ferroptosis. To further confirm the occurrence of oxidative damage in fungal biofilms, the lipid peroxidation probe BODIPY 581 / 591C11 was used. Figure 29As shown, fluorescence probe detection showed that after incubation of the antibiotic nanozyme at 1 mg / mL with fungi, the level of lipid peroxide increased significantly, demonstrating the occurrence of fungal membrane peroxidation during the anti-drug-resistant Candida albicans effect of the antibiotic nanozyme. Meanwhile, an increase in lipid peroxide was also observed in the fungi in the fluconazole treatment group, which may be related to the effect of fluconazole on damaging the cell membrane.

[0104] (4) The increased level of fungal lipid peroxidation after treatment with the fluconazole nanozyme was detected by directly detecting the lipid peroxidation product malondialdehyde (MDA). As Figure 30 shown, the MDA content in the drug-resistant Candida albicans cells in the 1 mg / mL fluconazole nanozyme treatment group increased significantly compared with the control group, indicating the occurrence of fungal lipid peroxidation and also proving that the peroxidase activity of the fluconazole nanozyme can cause lipid peroxidation in the fungal cells, which is an important mechanism involved in antibacterial action.

[0105] (5) During the ferroptosis process, the depletion of intracellular GSH will accelerate lipid peroxidation and cell membrane damage. Due to the peroxidase activity of the antibiotic nanozyme, the GSH content in the fungi treated with FCZ(Hemin) and CRO(Hemin) was detected to explore the possibility of promoting antibacterial action by reducing the fungal GSH level. As Figure 31 shown, the antibiotic nanozyme could significantly scavenge GSH, indicating that in the antibacterial action, the antibiotic nanozyme also exerted GSH-Px enzyme activity, which is another antibacterial mechanism of the enzyme catalytic activity of the antibiotic nanozyme in addition to ROS regulation.

[0106] (6) The surface morphology of drug-resistant Candida albicans after treatment with PBS was observed by scanning electron microscopy. The results are as Figure 32 shown. The surface structures of the untreated group (Control), the Hemin and FCZ treatment groups of Candida albicans were intact, the cell walls were intact and smooth, without obvious damage effects. In contrast, a large amount of material could be observed attached around the fungal cells in the FCZ(Hemin) treatment group, and the morphological structure of Candida albicans changed significantly (showing a porous network structure), and the cell wall was severely damaged. It indicates that the antibiotic nanozyme had a destructive effect on the fungal membrane.

[0107] (7) The above results showed that the interaction between the antibiotic nanozyme and fungi could induce ferroptosis in fungi, and its biological characteristics included increased ROS, lipid peroxidation, cell membrane contraction, GSH consumption and gene levels, etc. To verify the occurrence of ferroptosis, the typical ferroptosis inhibitor Ferrostatin-1 was added to the mixture of the antibiotic nanozyme and fungi, and the antibacterial activity was detected by measuring the absorbance at OD 600 nm. The ferroptosis inducer was also evaluated using the same model. The results are as Figure 33As shown. The results showed that after co-treatment with heme, the assembled antibiotic nanozyme could reverse the resistance of drug-resistant Candida albicans to fluconazole and ceftriaxone sodium. These results all provided evidence for the direct induction of ferroptosis in fungi by antibiotic nanozymes.

[0108] (8) To further understand the antifungal mechanism of the antibiotic assembly, we analyzed the fungi treated with FCZ and FCZ(Hemin) by RNA-seq. The Volcano plots of differentially expressed genes are as Figure 34 shown. We found that after treatment with FCZ(Hemin), the drug-resistant genes of Candida albicans to fluconazole, such as MRR1, TAC1, CDR, CDR2, etc., were all down-regulated, while they were up-regulated in the FCZ group. And these genes are all involved in encoding efflux pumps. It may be because FCZ activated these efflux pump-related genes, while FCZ(Hemin) can reduce the overexpression of these drug-resistant genes. Then, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were used to further evaluate the antibacterial activity of FCZ(Hemin). The KEGG enrichment plots of FCZ and FCZ(Hemin) are respectively as Figure 35 and Figure 36 shown. The ABC transporter pathway was significantly enriched, and the protein encoded by CDR1 is an ABC transporter, which uses ATP hydrolysis to provide energy to actively transport drugs such as fluconazole from inside the cell to outside the cell. This efflux reduces the effective concentration of the drug inside the cell, weakens the inhibitory effect of the drug on fungi, and thus enhances drug resistance.

[0109] The results of differential expression analysis of ferroptosis-related genes and genes encoding efflux pumps in fungi are respectively as Figure 37 and 38 shown. Transcriptome analysis showed that compared with untreated and FCZ-treated Candida albicans, the expression of GPX1 and GPX2 in fungi in the FCZ(Hemin) treatment group was up-regulated. GPX1 and GPX2 are core molecules of the antioxidant defense system, responsible for scavenging lipid peroxides to protect cells from ferroptosis. In fungi treated with fluconazole nanozyme, the expression of GPX1 and GPX2 increased.

[0110] FTH1 is the heavy chain subunit of ferritin. In iron metabolism, ferritin plays a role in sequestering and storing iron ions, preventing iron ions from catalyzing oxidation reactions, and thus avoiding oxidative stress. In fungi treated with fluconazole, the expression of the ferroptosis-related gene FTH1 increased.

[0111] SOD3 scavenges specific types of ROS through its encoded superoxide dismutase 3, thereby reducing the level of oxidative stress. Thus, it may affect the key trigger factor of ferroptosis - iron-catalyzed lipid peroxidation reaction.

[0112] Experimental Example 7

[0113] The safety of the antibiotic nanozyme was evaluated by a hemolysis test. Blood was collected from the orbital cavity of mice into anticoagulant tubes, and then 25 μL of whole blood was added to 1 mL of FCZ (Hemin) at different concentrations (0 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, 500 μg / mL, and 1000 μg / mL). At the same time, ultrapure water (1 mL) and PBS (1 mL) were used as positive and negative controls, respectively. After standing for 30 min, centrifugation was performed at 3000 g and 4 °C for 5 min. 100 μL of the supernatant was taken, and an L-lactate dehydrogenase (L-LDH) activity detection kit was used to detect the content of L-LDH in the serum of each group to reflect the hemolysis of the samples. The results are as Figure 39 shown, and it was found that the antibiotic nanozyme has good blood compatibility.

[0114] To evaluate the in vivo biocompatibility of the antibiotic nanozyme, healthy Balb / c mice were treated with different concentrations of the antibiotic nanozyme, and the effects on body weight, blood routine, biochemical indicators, and major organs were observed. The results are as Figure 40-42 shown. Compared with the PBS group, there were no significant differences in the above indicators in the antibiotic nanozyme group. These results all indicate that the antibiotic nanozyme has good in vivo biocompatibility.

[0115] Experimental Example 8

[0116] The anti-drug-resistant bacteria effect of fluconazole nanozyme in organisms was verified through the treatment of a mouse epidermal wound infection model (as Figure 43 shown). To evaluate the anti-infective effect of FCZ (Hemin), a mouse wound infection model was established. After anesthetizing the mice by intraperitoneal injection of 200 μL of sodium pentobarbital (10 mg / mL), hair removal and disinfection were performed on the back. After creating a wound with a sterile scalpel, 20 μL of an activated drug-resistant Candida albicans fungal suspension (about 1×108 CFU mL-1) was dropped on the wound to infect the wound surface, and the infected model mice were randomly divided into 4 groups (n = 6). 24 h after infection, the wounds of each group of mice were administered according to the grouped doses, and were treated with control (PBS), FCZ (1 mg mL-1), Hemin (2 mg mL-1), and FCZ (Hemin) (3 mg mL-1) respectively. The administration volume was 20 μL. The wounds of each group were treated and photographed on days 1, 3, 5, 7, and 9 respectively until euthanasia. 10 days after treatment, for in vivo antifungal analysis, the wound homogenate was serially diluted, and the diluted homogenate was spread on SDB plates and incubated at 37 °C for 48 h, and then colony counting was performed to calculate log10 (CFU of each mouse's wound). ImageJ software was used to quantify the data of the wound images. The healing of the infected wound surface of the mice is as Figure 44As shown, the relative area percentage and healing rate of the infected wounds of the mice are as follows Figure 45 shown

[0117] It can be seen from Figure 44-45 that after 5 treatments, on the 9th day of the experiment, the skin tissue of the fluconazole nanozyme group had basically fully recovered and recovered better than the other groups.

[0118] Then, the fungal burden of the wounds of each group of mice was further measured. After grinding and plating the wound tissues, fluconazole did not reduce the bacterial burden below the initial infection level. As Figure 46 shown, the antibiotic nanozyme showed strong antibacterial activity. Compared with the control group, the colony-forming units (CFUs) decreased by more than 2 log10. It was shown that the fluconazole nanozyme played a good activity in clearing drug-resistant Candida albicans in the infected mouse epidermal wounds, promoting the healing and repair of the wounds.

[0119] To further evaluate the antibacterial effects of CRO (Hemin) and FCZ (Hemin) in a mouse pneumonia infection model, we constructed a mouse pneumonia model. The bacterial solution in the logarithmic growth phase was induced to form hyphae, and then the mice were anesthetized with isoflurane and inoculated with the pre-induced bacterial solution through the nasal cavity. The mice were respectively intranasally instilled with 100 μL of drug-loaded (0.9% normal saline), CRO (20 mg / kg), CRO (40 mg / kg), CRO (Hemin) (30 mg / kg), CRO (Hemin) (60 mg / kg), and FCZ (Hemin) (>44 mg / kg) on days 1, 3, 5, and 7 after infection. Nine days after treatment, the mice were euthanized by cervical dislocation, and the heart, liver, spleen, lungs, and kidneys were taken for histopathological evaluation. The lung tissues were collected to measure the fungal burden. The lung tissue homogenate was serially diluted on an SDB agar plate and incubated at 37 °C for 24 - 48 h, and then colony counting was performed. The detection results of the fungal burden in the mouse lung tissues are as Figure 47 shown. The results showed that both CRO (Hemin) and FCZ (Hemin) could effectively kill the fungi in the mouse lungs, and the effect was better than that of other groups (more than 2 Log more) at the same mass concentration. After the treatment of pneumonia mice, as shown by H&E Figure 48 shown, in the FCZ (Hemin) treatment group, the alveolar septa of the mice were normal, and there were no obvious pathological changes in the alveolar cavity except for normal alveolar epithelial cells. However, a large number of inflammatory cells were infiltrated in the alveolar interstitium of the mice in the PBS, Hemin, and FCZ groups, such as an increase in the number and density of lymphocytes and phagocytes.

[0120] Finally, to further evaluate the potential of antibiotic assemblies as topical antifungal agents, we evaluated their antifungal effects against Candida albicans-induced vaginitis in vivo. Since Candida does not exist in the natural murine vaginal microecosystem, a pathological state conducive to vaginal fungal infection needs to be created by hormones. To maintain the persistent state of Candida albicans infection in mice, a pseudoproliferative state of female mice was established by injecting estrogen throughout the experiment, including before fungal inoculation. Then, a VVC model was established by inoculating the vagina with a Candida albicans suspension, and fungal infection was confirmed by culturing the growth of Candida albicans from the vaginal secretions of infected animals. After the treatment was completed, vaginal lavage fluid was taken for plating, photographing, and counting. The results are as Figure 49 shown. After treatment with CRO(Hemin), the Candida albicans colonization in the vaginal area decreased by approximately 1 log10 compared to the control group, and FCZ(Hemin) decreased by more than 2 log10.

[0121] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, or improvements made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. An antibiotic nanozyme that reverses drug resistance, characterized in that: Assembled from antibiotics and hemin.

2. The antibiotic nanozyme for reversing drug resistance according to claim 1, characterized in that The antibiotic is at least one of fluconazole, vancomycin, ceftriaxone sodium, ampicillin, azithromycin, novobiocin, cephalexin and gentamicin.

3. The antibiotic nanozyme for reversing drug resistance according to claim 1, characterized in that The preparation method comprises the following steps: (1) dissolving hemin chloride in dimethyl sulfoxide to obtain a hemin solution; (2) dissolving the antibiotic in ultrapure water to obtain an antibiotic solution; (3) The heme solution obtained in step (1) is mixed with the antibiotic solution obtained in step (2), and then the pH is adjusted to 6.5-7.5 with sodium hydroxide solution. After sealing, the mixture is stirred overnight at room temperature, and then centrifuged and freeze-dried in a vacuum to obtain an antibiotic nanozyme that reverses drug resistance.

4. The antibiotic nanozyme for reversing drug resistance according to claim 3, characterized in that In step (3) of the preparation method, the concentration of the heme solution is 0.04-0.06 mM / mL.

5. The antibiotic nanozyme for reversing drug resistance according to claim 3, characterized in that In step (3) of the preparation method, the concentration of the antibiotic solution is 0.08-0.12 mM / mL.

6. The antibiotic nanozyme for reversing drug resistance according to claim 3, characterized in that In step (3) of the preparation method, the volume ratio of the heme solution to the antibiotic solution is 1:0.8-1.

2.

7. The antibiotic nanozyme for reversing drug resistance according to claim 3, characterized in that In step (3) of the preparation method, the concentration of the sodium hydroxide solution is 0.8-1.2 mol / L.

Images