A nanocomposite and its application

Abstract

This application relates to a nanomolecular complex comprising: a zeolite imidazolate framework (ZIF-8); and pyrithione (PT); wherein the pyrithione (PT) is supported on the ZIF-8 framework. The nanoantimicrobial agent of this application uses nanomaterials as carriers and incorporates a dual antimicrobial mechanism to target bacterial infections and other sites. While exhibiting highly effective antimicrobial effects, it does not induce bacterial resistance and can significantly reduce or even eliminate the development of superbugs.

Description

Technical Field

[0001] The present application relates to the preparation and application of antibacterial agents, and in particular to a nanocomposite and its application. Background Art

[0002] Bacterial infections seriously threaten human health, and antibiotics are often used for treatment in clinical and actual production applications. However, due to the effects of body metabolism and biofilm barriers, it is difficult for antibiotics to enrich and kill intracellular bacteria at the site of infection. In the past, people often achieved therapeutic effects by increasing the dosage of antibiotics, but the abuse of antibiotics can cause serious bacterial resistance and even cause super bacteria to wreak havoc, resulting in a vicious cycle (RA Fisher, B. Gollan, S. Helaine, Persistent bacterial infections and persister cells, Nat Rev Microbiol 15(8)(2017)453-464.;E. Christaki, M. Marcou, A. Tofarides, Antimicrobial Resistance in Bacteria: Mechanisms, Evolution, and Persistence, J Mol Evol 88(1)(2020)26-40.).

[0003] Meanwhile, abdominal infections are the most common complication of modern surgery caused by drug-resistant bacteria (Nicoletti et al., 2009), which can lead to acute infection, inflammation, and even death. Antibiotics are the most commonly used drugs for preventing infection, but they can also cause drug resistance (Sartelli et al., 2017). The search for new treatments for abdominal infections is urgent.

[0004] Application Contents

[0005] In response to the technical problems existing in the prior art, the present application proposes a nanomolecular complex, comprising: a zeolite imidazolate skeleton ZIF-8; and pyridinethione (PT); wherein the pyridinethione (PT) is loaded on the zeolite imidazolate skeleton ZIF-8.

[0006] The nanomolecular complex as described above, wherein the molar mass ratio of ZIF-8 to PT is (8-15):10.

[0007] The nanomolecular complex as described above, wherein the mass ratio of ZIF-8 to PT is (0.5-3.5):1.

[0008] The nanomolecular complex as described above, wherein the zeolite imidazolate framework ZIF-8 comprises zinc sulfate (ZnSO4) and 2-methylimidazole, which are formed in an aqueous solution; wherein Zn 2+ , 2-methylimidazole and water has a molar ratio of 1: (65-75): (1000-1500).

[0009] The nanomolecular complex as described above has a size of (180-210) nm, preferably 193.80±2.68 nm; and a zeta potential of (15-40) mV, preferably -27.80±4.98 mV.

[0010] A nano antibacterial agent comprises any one of the above nano molecular complexes.

[0011] Use of any of the above-mentioned nanomolecular complexes in the preparation of nano antibacterial agents for preventing and / or treating related diseases caused by pathogens.

[0012] In the application described above, the nano antibacterial agent is a pH-responsive antibacterial agent.

[0013] The application as described above includes applying an effective dose of nano-antibacterial agent to pathogens, and the effective application dosage is a concentration of the nano-complex of not less than 3μg / ml; preferably, the effective dose for inhibiting the growth of pathogens is a concentration of the nano-complex of not less than 3.5μg / ml, preferably not less than 4μg / ml; preferably, the effective dose for killing pathogens is a concentration of the nano-complex of not less than 7μg / ml, preferably not less than 8μg / ml.

[0014] Application of any of the above-mentioned nanomolecular complexes or the above-mentioned nanoantibacterial agents in influencing the morphology of pathogen biofilms.

[0015] As described above, the nanomolecular complex or nano antibacterial agent affects the morphology of pathogen biofilm by inhibiting the formation of biofilm by pathogens or clearing the biofilm already formed by pathogens.

[0016] As described above, the nanomolecular complex or nano antibacterial agent affects the morphology of pathogen biofilm by causing the pathogen surface to collapse, shrink and / or rupture.

[0017] In any of the above applications, the pathogen is Aeromonas vernix.

[0018] A method for killing or inhibiting the growth of pathogens in vitro, comprising administering an effective dose of any one of the above nanocomplexes or the above nano antibacterial agents to the pathogens.

[0019] Use of any of the above-mentioned nanocomplexes or nano antibacterial agents in the preparation of drugs for preventing and / or treating pathogen infections in fish or mammals.

[0020] In the application as described above, the infection is an abdominal infection.

[0021] As described above, the mammals include but are not limited to humans, primates, rodents, rabbits, dogs, and livestock.

[0022] As described in 7 above, the mammals include but are not limited to humans, chimpanzees, monkeys, mice, rats, rabbits, dogs, pigs, cattle, and sheep.

[0023] The nano antibacterial agent of the present application uses nanomaterials as carriers and combines dual antibacterial mechanisms to target sites of bacterial infection. While having a highly effective antibacterial effect, it will not cause bacteria to develop drug resistance and can also significantly reduce or even eliminate the generation of super bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Below, the preferred embodiments of the present application will be further described in detail with reference to the accompanying drawings, wherein:

[0025] Figure 1 1 is a characterization diagram of ZIF-8 and ZIF-8@PT according to an embodiment of the present application, wherein (a) is a characterization diagram under a scanning electron microscope; (b) is a characterization diagram under a transmission electron microscope;

[0026] Figure 2 1 is a characterization diagram of ZIF-8 and ZIF-8@PT according to another embodiment of the present application, wherein A is a particle size histogram of ZIF-8 and ZIF-8@PT; B is a potential histogram of ZIF-8 and ZIF-8@PT;

[0027] Figure 3 is a pH-responsive release curve of ZIF-8@PT according to one embodiment of the present application;

[0028] Figure 4 The anti-Aeromonas vinblastini effect of ZIF-8@PT under different pH conditions and different treatment times according to one embodiment of the present application is shown in FIG. Figure 4 (a) is a graphical representation of the survival and growth of Aeromonas vernix treated with PBS under different pH conditions according to one embodiment of the present application; Figure 4 (b) is a graphical representation of the survival and growth of Aeromonas vernix treated with ZIF-8@PT under different pH conditions according to one embodiment of the present application; Figure 4 (c) is a bar graph showing the survival of Aeromonas vernix treated with PBS under different pH conditions according to one embodiment of the present application; and Figure 4(d) is a bar chart showing the survival of Aeromonas vermiformis treated with ZIF-8@PT under different pH conditions according to one embodiment of the present application;

[0029] Figure 5 The effect of ZIF@PT on the biofilm, morphology and intracellular structure of Aeromonas veseri according to one embodiment of the present application is shown; wherein, Figure 5 (a) The inhibitory effect of ZIF-8@PT on Aeromonas vernix biofilm formation according to one embodiment of the present application; Figure 5 (b) shows the removal effect of ZIF-8@PT on an established Aeromonas vickers biofilm according to one embodiment of the present application; Figure 5 (c) is a scanning electron microscopy (SEM) image of Aeromonas vernix treated with ZIF-8 and PBS for 24 hours according to one embodiment of the present application; Figure 5 (d) is a transmission electron microscopy (TEM) image of Aeromonas vernix treated with ZIF-8, PT, and ZIF-8@PT for 24 h according to one embodiment of the present application;

[0030] Figure 6 This is the experimental design and results of ZIF-8@PT inhibiting abdominal infection according to one embodiment of the present application; wherein, Figure 6 (a) A treatment experiment design for Aeromonas vernix infection according to one embodiment of the present application; Figure 6 (b) is a bacterial culture agar plate in the main organ tissues of mice after treatment with PBS, PT, ZIF-8@PT and kanamycin (Kan) according to one embodiment of the present application; Figure 6 (c) shows the bacterial counts in the hearts of mice treated with PBS, PT, ZIF-8@PT, and kanamycin (Kan) according to an embodiment of the present application; Figure 6 (d) shows the bacterial counts in the liver of mice treated with PBS, PT, ZIF-8@PT, and kanamycin (Kan) according to an embodiment of the present application; Figure 6 (e) shows the bacterial counts in the spleen of mice treated with PBS, PT, ZIF-8@PT, and kanamycin (Kan) according to an embodiment of the present application; Figure 6 (f) shows the bacterial counts in the lungs of mice treated with PBS, PT, ZIF-8@PT, and kanamycin (Kan) according to an embodiment of the present application; Figure 6 (g) shows the bacterial counts in the kidneys of mice treated with PBS, PT, ZIF-8@PT, and kanamycin (Kan) according to an embodiment of the present application; Figure 6 (h) is the survival rate of infected mice after treatment with PBS, PT, ZIF@PT and kanamycin (Kan) according to one embodiment of the present application; Figure 6(i) is the level of IL-4 in the serum of the control group and infected mice after treatment according to one embodiment of the present application; Figure 6 (j) is the level of IL-6 in the serum of the control group and infected mice after treatment according to one embodiment of the present application; and Figure 6 (k) is the level of IL-1β in the serum of the control group and infected mice after treatment according to one embodiment of the present application; Figure 6 Medium, N = 5, ***p < 0.01, ***p < 0.001, ****p < 0.0001; and

[0031] Figure 7 This is a test of the drug resistance of Aeromonas vermifuge to ZIF-8@PT according to one embodiment of the present application. DETAILED DESCRIPTION

[0032] To make the purpose, technical solutions, and advantages of the embodiments of this application more clear, the technical solutions in the embodiments of this application will be clearly and completely described below in conjunction with the drawings in the embodiments of this application. Obviously, the described embodiments are part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0033] In the detailed description that follows, reference is made to the accompanying drawings that form part of this application and illustrate specific embodiments of the present application. In the accompanying drawings, similar reference numerals describe substantially similar components in different figures. Each specific embodiment of the present application is described below in sufficient detail to enable a person of ordinary skill in the art to implement the technical solutions of the present application. It should be understood that other embodiments may be utilized or structural changes may be made to the embodiments of the present application.

[0034] Bacterial infections pose a serious threat to human health. When antibiotics are used to treat bacterial infections, it is difficult for antibiotics to accumulate at the site of infection, and even more difficult to kill intracellular bacteria, due to the effects of body metabolism and biofilm barriers. Excessive use of antibiotics to achieve good antibacterial effects will lead to serious bacterial resistance and even cause superbugs to wreak havoc. The nano-antibacterial agent of the present application uses nanomaterials as carriers and combines dual antibacterial mechanisms to target sites of bacterial infection. While having a highly effective antibacterial effect, it will not cause bacteria to develop drug resistance and can significantly reduce or even eliminate the generation of superbugs.

[0035] The terms used in this application have the following meanings:

[0036] The "nanocomplex" mentioned herein refers to the ZIF-8@PT complex formed after pyrithione (PT) is loaded on the zeolite imidazolate framework ZIF-8. In some embodiments, the size of the nanocomplex is 193.80±2.68nm and the zeta potential is -27.80±4.98mV. In some embodiments, ZIF-8@PT can inhibit the formation of pathogen biofilms and eliminate biofilms that have already formed. In some embodiments, after ZIF-8@PT treats pathogens, collapse, shrinkage and some ruptures can be clearly observed on the surface of the pathogens. In some embodiments, the pathogen is a pathogenic bacterium, and further, the pathogen can be a drug-resistant pathogen. In some embodiments, the pathogen is Aeromonas vernix.

[0037] Furthermore, in some embodiments, the nanocomplex has a pH-responsive effect and is a targeted nanocomplex. Specifically, the nanocomplex can be targeted to inflammatory sites in acidic environments or sites where pathogens accumulate, inhibiting or killing pathogens.

[0038] Furthermore, the present invention synthesizes ZIF-8 via a simple co-precipitation method. A certain concentration of PT is then mixed with ZIF-8, shaken at room temperature for 24 hours, and centrifuged to dry to obtain ZIF-8@PT crystals. In some embodiments, the ZIF-8-loaded PT is highest after 24 hours of shaking at room temperature, reaching 36.5%.

[0039] "Pyrithione" or "PT" as used herein refers to N-oxide-2-thiopyridine (pyrithione, PT), a metal binder that has been shown to have broad-spectrum antifungal and antibacterial activity (GLDiamond, NP Skoulis, AR Jeffcoat, JF Nash, A Physiological-Based Pharmacokinetic Model For The Broad Spectrum Antimicrobial Zinc Pyrithione: II. Dermal Absorption And Dosimetry In The Rat, J Toxicol Environ Health A 84(15)(2021)609-631.). Among them, zinc pyrithione (ZPT), formed with metallic zinc, is the most widely used anti-dandruff ingredient with broad-spectrum and highly effective antibacterial and antifungal properties.

[0040] The "ZIF-8" mentioned in this article is a zeolitic imidazolate framework, a typical representative of ZIFs. It is formed by the coordination of metallic zinc and dimethylimidazole and can be used as a drug delivery carrier. Existing literature records that drug carriers based on nanomaterials can overcome the biofilm barrier and deliver antibiotics to the cytoplasm to kill intracellular bacteria, thereby reducing the amount of antibiotics used. They also have the advantages of low drug resistance, broad spectrum, and targeting, making them ideal carriers for delivering antibiotics (W.Gao, L.Zhang, Nanomaterials arising amid antibiotic resistance, Nat Rev Microbiol 19(1)(2021)5-6.). Among them, zeolitic imidazolate frameworks (ZIFs) are relatively popular nanomaterials in recent years. They have excellent properties such as good pore size, adjustable structure and composition, adjustable size, wide range of functions, high drug loading capacity, and good biocompatibility. They are very promising drug delivery carriers (J. Yang, YW Yang, Metal-Organic Frameworks for Biomedical Applications, Small 16(10)(2020)e1906846.; J. Troyano, A. Carne-Sanchez, C. Avci, I. Imaz, D. Maspoch, Colloidal metal-organic framework particles: the pioneering case of ZIF-8, Chem Soc Rev 48(23)(2019)

[0041] 5534-5546.).

[0042] Since zinc ions themselves have antibacterial effects, the drug delivery system based on ZIF-8 framework can improve the antibacterial efficiency of drugs (Y.Su, I.Cockerill, Y.Wang, Y.Qin, L.Chang, Y.Zheng, DJTibZhu, Zinc-Based Biomaterials for Regeneration and Therapy, 37(4)(2019)

[0043] 428-441.). And the advantage of its pH responsiveness has also received widespread attention and application. Since the drug-carrying system is mainly targeted at the acidic environment of the tumor site, bacteria or membrane, ZIF-8 has a targeting effect as a loading material. In a biological environment (pH is neutral), the structural stability can be maintained to prevent drug release as much as possible. When the drug reaches the lesion site, it releases the drug due to pH response, which can improve the bioavailability of the drug and reduce the toxic and side effects of the drug (X.Mi, M.Hu, M.Dong, Z.Yang, X.Zhan, X.Chang, J.Lu, XJIjonChen, Folic Acid Decorated Zeolitic Imidazolate Framework (ZIF-8) Loaded with Baicalin as a Nano-Drug Delivery System for Breast Cancer Therapy, 16 (2021) 8337-8352.).

[0044] The "antimicrobial agent" referred to herein refers to an antimicrobial agent comprising the aforementioned nanocomposite ZIF-8@PT complex, which has the ability to inhibit and kill pathogens, such as pathogenic bacteria, including drug-resistant bacteria. In some embodiments, the antimicrobial agent can be a liquid preparation, lyophilized preparation, powder, tablet, capsule, etc. comprising the aforementioned nanocomposite.

[0045] The "dual antibacterial mechanism" or "dual antibacterial" mentioned herein refers to the prodrug PT loaded into ZIF-8, which can target the acidic site of bacterial infection and release PT and zinc ions at the site. 2+ Self-assembly forms ZPT. Both PT and ZPT possess antibacterial properties, effectively enhancing antibacterial efficacy. In some embodiments, the nanoantimicrobial agent containing the ZIF-8@PT complex exhibited a minimum inhibitory concentration of 4 μg / ml against Aeromonas victoriae, a 32-fold decrease compared to PT alone. The minimum bactericidal concentration was 8 μg / ml, achieving complete kill of Aeromonas victoriae in just four hours.

[0046] Aeromonas vickers, as described in this article, belongs to the genus Aeromonas in the family Vibrio. It is a Gram-negative, facultatively anaerobic bacillus. It is widely found in various aquatic ecosystems and is a common pathogen in humans and fish. It is frequently isolated from human feces with diarrhea and fish with hemorrhagic septicemia.

[0047] The terms "drug resistance" and "drug resistance" refer to the tolerance of bacteria to the effects of antimicrobial drugs. Once drug resistance occurs, the antimicrobial effect of the drug will be significantly reduced. Drug resistance can be divided into acquired resistance and natural resistance according to the cause of its occurrence. Pathogens in nature, such as a certain strain of bacteria, may also have natural drug resistance. When antibiotics are used for a long time, the majority of sensitive strains are continuously killed, and the resistant strains multiply in large numbers, replacing the sensitive strains, and the resistance rate of bacteria to the drug continues to increase. In the present application, the inventors have conducted a large number of experiments for more than a month and have not found any resistance characteristics of pathogens to the nano antimicrobial agent of the present application.

[0048] The nanocomposite of the present application is obtained by loading PT on ZIF-8, wherein the molar mass ratio of ZIF-8 to PT in ZIF-8@PT is (8-15):10. Further, in some embodiments, the molar mass ratio of ZIF-8 to PT in ZIF-8@PT is 8:10, or 9:10, or 10:10, or 11:10, or 12:10, or 13:10, or 14:10, or 15:10. In some embodiments, the molar mass ratio of ZIF-8 to PT in ZIF-8@PT is not an integer ratio, such as the molar mass ratio of ZIF-8 to PT in ZIF-8@PT is 8.1:10, or 8.2:10 ... or 14.8:10, or 14.9:10, and so on, which are not listed here one by one. Or the mass ratio of ZIF-8 to PT in ZIF-8@PT is (0.5-3.5):1. Further, in some embodiments, further, the mass ratio of ZIF-8 to PT in ZIF-8@PT is 0.5:1, or 0.6:1, or 0.7:1, or 0.8:1, or 0.9:1, or 1:1, or 1.1:1, or 1.2:1, or 1.3:1, or 1.4:1, or 1.5:1, or 1.6:1, or 1.7:1, or 1.8:1, or 1.9:1, or 2.0:1, or 2.1:1, or 2.2:1, or 2.3:1, or 2.4:1, or 2.5:1, or 2.6:1, or 2.7:1, or 2.8:1, or 2.9:1, or 3.0:1, or 3.1:1, or 3.2:1, or 3.3:1, or 3.4:1, or 3.5:1, etc. Those skilled in the art should understand that the above-mentioned molar mass ratios and mass ratios do not limit the molar mass ratios and mass ratios of the present application, but are merely examples within the scope of the above-mentioned molar mass ratios and mass ratios.

[0049] ZIF-8 is synthesized by mixing 2-methylimidazole solution with zinc sulfate solution. 2+, 2-methylimidazole and H2O has a molar ratio of 1:(65-75):(1000-1500); further, the molar ratio is 1:70:1238.

[0050] In one embodiment, the size of ZIF-8@PT is (180-210) nm. Further, in some embodiments, the size of ZIF-8@PT is 193.80±2.68 nm. In some embodiments, the zeta potential of ZIF-8@PT is (15-40) mV; further, the zeta potential of ZIF-8@PT is -27.80±4.98 mV.

[0051] The nanocomplex of the present application has significant antibacterial activity against pathogens, such as Aeromonas vermiformis, and does not screen out drug-resistant strains. In some embodiments, the nano-antibacterial agent of the present application can be used to prevent and treat pathogen infections in fish, primates, rodents, rabbits, canines, livestock, and even humans, such as treating pathogen infections in fish, humans, chimpanzees, monkeys, mice, rats, rabbits, dogs, pigs, cattle, and sheep. Furthermore, according to one embodiment of the present application, the nano-antibacterial agent of the present application can be used to treat or prevent abdominal pathogen infections caused by animal surgery, etc.

[0052] In some embodiments, when the nano-antimicrobial agents of the present application are used to prevent or treat pathogen infections in animals, the administration method may be injection, oral administration, etc. In some embodiments, the nano-antimicrobial agents of the present application may be in the form of liquid preparations, lyophilized preparations, powders, tablets, capsules, etc. In particular, when the nano-antimicrobial agents of the present application are used to prevent and treat pathogen infections in fish, the nano-antimicrobial agents may be dissolved in an aqueous environment or mixed into aquaculture feed.

[0053] Example 1 Preparation of ZIF-8 and ZIF-8@PT

[0054] In this example, ZIF-8 was synthesized by a coprecipitation method. The specific steps include: first, dissolving 1.242 g of zinc sulfate hexahydrate in 8 ml of deionized water. Second, dissolving 22.70 g of 2-methylimidazole in another 80 ml of water. The zinc sulfate solution and the 2-methylimidazole solution were then stirred and mixed at room temperature. After the two solutions were mixed, the resulting solution turned milky white almost immediately. After stirring at room temperature for approximately 5 minutes, the mixture was centrifuged at 9000 rpm for 5 minutes. The unreacted substances were removed by washing with deionized water several times, and the product was dried in a vacuum oven at 55°C for 12 hours to produce ZIF-8.

[0055] PT and ZIF-8 were weighed in a ratio of 1:2 and added to 10 mL of distilled water for ultrasonic dispersion. The mixture was adsorbed at room temperature for 24 h, centrifuged at 5000 r / min for 10 min, washed three times with 10 mL of distilled water, and dried in an oven at 60°C for 12 h to obtain ZIF-8@PT crystals.

[0056] Among them, according to the inventor's multiple experiments and the determination of the PT loading on ZIF-8 after shaking for different times, it can be found that the amount of PT loaded on ZIF-8 after shaking for 24 hours is the highest, up to 36.5%. In one embodiment, the size of ZIF-8@PT is 193.80±2.68nm, and the zeta potential is -27.80±4.98mV. In some embodiments, the molar mass ratio of ZIF-8 to PT in ZIF-8@PT crystals is 11:10; or in some embodiments, the mass ratio of ZIF-8 to PT in ZIF-8@PT is 2:1. ZIF-8 is synthesized by mixing 2-methylimidazole solution with zinc sulfate solution, and Zn in the solution is 0.05. 2+ , 2-methylimidazole and H2O has a molar ratio of 1:(65-75):(1000-1500); further, the molar ratio is 1:70:1238.

[0057] Figure 1 Schematic representation of ZIF-8 and ZIF-8 loaded with PT according to one embodiment of the present application; wherein Figure 1 (a) is a scanning electron microscope image; and Figure 1 (b) is a characterization diagram under a transmission electron microscope. Figure 1 (a) and Figure 1 As shown in (b), the loading of PT affects the structure of ZIF. Furthermore, loading of PT does not affect the function of ZIF.

[0058] Figure 2 This is a characterization diagram of ZIF-8 and ZIF-8@PT according to another embodiment of the present application, wherein A is a particle size histogram of ZIF-8 and ZIF-8@PT; B is a potential histogram of ZIF-8 and ZIF-8@PT. Figure 2 A and Figure 2 As shown in Figure B, the particle size of ZIF-8 did not change much before and after loading with PT, and the potential of ZIF-8 changed from -25.4 mV before loading to -27.8 mV.

[0059] Example 2 pH-responsive release analysis of ZIF-8@PT

[0060] Previous work investigated the pH-responsiveness of ZIF-8@PT. Briefly, the release of PT from ZIF-8@PT at different pH levels was investigated as follows: 10 mg of ZIF-8@PT was dispersed in 20 mL of 0.5% (v / v) Tween 20 / PBS solution and incubated at 37°C with constant agitation on a shaker at 150 rpm. One mL of the supernatant was then added to 1 mL of buffer at different pH values at various time points (0.5, 1, 2, 4, 8, 16, 32, and 64 hours). The supernatant was then diluted 20-fold with ethanol, and the absorbance was measured at 362 nm. The PT release was calculated using a standard curve.

[0061] Figure 3 This is the pH response release curve of ZIF-8@PT according to one embodiment of the present application. Figure 3 As shown in the Figure 3, the release of PT from ZIF-8@PT is pH-dependent. In an acidic environment with a pH of 5.5, the amount of PT released is much higher than that in a neutral environment with a pH of 7.4.

[0062] Example 3 Antibacterial activity of ZIF-8@PT against Aeromonas vernix

[0063] The minimum inhibitory concentration (MIC) of ZIF-8, PT, and ZIF-8@PT against Aeromonas vernix was determined using a microdilution method. 95 μL of A. vernix (1×106 colony-forming units (CFU) / mL) and 5 μL of ZIF-8@PT (512, 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5, and 0.25 μg / mL) were added to a 96-well plate. 5 μL of PBS was used as a control. Three replicates were run in parallel in each group, and the 96-well plate was incubated at 30°C for 24 hours. The absorbance of the 96-well plate was then measured at 600 nm using a microplate reader.

[0064] The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of ZIF-8, PT, and ZIF-8@PT against Aeromonas vernix were determined using the agar diffusion method. A 50 μL aliquot of the test bacterial suspension cultured in a 96-well plate was plated onto the agar. The number of colonies on each plate was counted, and the MBC was then determined.

[0065] The minimum inhibitory concentration (MIC) of ZIF-8, PT, and ZIF-8@PT against Aeromonas vernix was determined using the doubling microdilution method. 95 μL of a 0.01 OD bacterial solution was pre-added to a 96-well culture plate. In the experimental group, 5 μL of ZIF-8@PT was added to each well to achieve final concentrations of 512, 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5, and 0.25 μg / mL, respectively. Three replicate wells were set up in each group, and the plates were thoroughly mixed. The plates were then incubated at 30°C for 24 hours. The OD600 values were measured using a microplate reader to determine the MIC of the compounds.

[0066] The minimum bactericidal concentration (MBC) of the compound against Aeromonas vernix was determined using the agar diffusion method. From the bacterial suspension cultured in the 96-well plate, 50 μl of the test bacterial suspension from each tube showing no visible bacterial growth was transferred to drug-free LB agar plates. After overnight incubation, the number of colonies growing on each plate was less than 5, which was the minimum bactericidal concentration (MBC) of the drug.

[0067] Figure 4 (a)- Figure 4 (b) shows the antibacterial and bactericidal properties of ZIF-8@PT against Aeromonas victoriae. Table 1 shows the antibacterial properties of ZIF-8@PT against Aeromonas victoriae according to one embodiment of the present application. As shown in Table 1, according to the inventors' experimental records, the minimum inhibitory concentration of ZIF-8@PT against Aeromonas victoriae is 4 μg / mL, which is 32 times lower than that of PT alone. The minimum bactericidal concentration is 8 μg / mL, at which Aeromonas victoriae can be completely killed in just 4 hours.

[0068] Table 1 Antibacterial activity of ZIF-8@PT against Aeromonas vernix

[0069] MIC (μg / mL) minimum inhibitory concentration MBC (μg / mL) sterilization ZIF-8 ＞512 ＞512 PT 16 64 ZIF-8@PT (PT content 0.5 μg / mL) 4 8

[0070] Example 4 Antibacterial activity of ZIF-8@PT

[0071] 1×10 of A. veronii C4 6 CFU were dispersed into 1 mL of 1×PBS / medium containing 8 μg of ZIF-8@PT and incubated at 30°C for 0, 0.5, 1, 2, and 4 h. 100 μL of the sample was spread on an agar plate at different time intervals and incubated at 30°C for 24 h.

[0072] Figure 4 The anti-Aeromonas vinblastini effect of ZIF-8@PT under different pH conditions and different treatment times according to one embodiment of the present application is shown in FIG. Figure 4 (a) is a graphical representation of the survival and growth of Aeromonas vernix on a PBS-treated culture medium under different pH conditions according to one embodiment of the present application; Figure 4 (b) is a diagram showing the survival and growth of Aeromonas vernix on a culture medium treated with ZIF-8@PT under different pH conditions according to one embodiment of the present application. Figure 4 (a)- Figure 4 As shown in (b), the growth of Aeromonas victoriae in the culture medium treated with ZIF-8@PT was significantly inhibited, indicating that ZIF-8 loaded with PT exhibited significant antibacterial activity. Furthermore, the antibacterial activity at pH 5.5 was better than that at pH 7.4.

[0073] Example 5 Antibacterial activity of ZIF-8@PT under different pH conditions

[0074] To study the effect of pH on the antibacterial activity of ZIF-8@PT, 1×10 6 CFU (Cellular Fungal Incubation Unit) was measured at 0, 0.5, 1, 2, and 4 h. 100 μL of bacteria was smeared on agar plates at different time intervals. After incubation at 30°C for 24 h, the plates were imaged using a gel imager and the number of bacteria was counted using Image J software. Each experiment was repeated three times.

[0075] Figure 4 The anti-Aeromonas vinblastini effect of ZIF-8@PT under different pH conditions and different treatment times according to one embodiment of the present application is shown in FIG. Figure 4 (c) is a bar graph showing the survival of Aeromonas vernix on a culture medium treated with PBS under different pH conditions according to one embodiment of the present application; and Figure 4 (d) is a bar chart showing the survival of Aeromonas victoriae on a culture medium treated with ZIF-8@PT under different pH conditions according to one embodiment of the present application. Table 2 shows the inhibition rate of Aeromonas victoriae on a culture medium treated with ZIF-8@PT at different pH values. Figure 4 (c)- Figure 4 As shown in Table 1, the growth of Aeromonas victoriae in the culture medium treated with ZIF-8@PT was significantly inhibited, indicating that ZIF-8 loaded with PT exhibited significant antibacterial activity. Furthermore, the antibacterial activity at pH 5.5 was better than that at pH 7.4.

[0076] Table 2 Inhibitory rate of ZIF-8@PT treated culture medium against Aeromonas victoriae

[0077]

[0078] Example 6 Effect of ZIF-8@PT on biofilm formation

[0079] Biofilms are powerful barriers that prevent antibiotics from entering bacteria and protect against environmental stress. Their formation is a key virulence factor in chronic infections caused by various microorganisms. Therefore, the ability to inhibit biofilm formation and remove established biofilms is essential for a good antimicrobial substance. (H.Koo, R.Allan, R.Howlin, P.Stoodley, LJNrMHall-Stoodley, Targeting microbial biofilms: current and prospective therapeutic strategies, 15(12)(2017)740-755.Z.Chen,Z.Wang,J.Ren,XJAocrQu,Enzyme Mimicry for Combating Bacteria and Biofilms,51(3)(2018)789-799.M.Valdes-Pena,N.Massaro,Y.Lin,JJAocrPierce,Leveraging MarineNatural Products as a Platform to Tackle Bacterial Resistance andPersistence,54(8)(2021)1866-1877.)

[0080] During the experiment, the experimenters added Aeromonas veronii C4 (1×10 6 CFU / mL) was added, and incubated at 30℃ for 36h. After washing with PBS three times, PBS and ZIF-8@PT 100μL were added and incubated for 24h. 100μL of Aeromonas veronii C4 (1×10 6 CFU / mL), PBS, and ZIF-8@PT (8 μg / mL). Incubate at 30°C for 36 hours. Dissolve 200 μL of crystal violet (1%) and incubate at room temperature for 10 minutes. Dissolve in 33% glacial acetic acid and record the absorbance at 590 nm using a microplate reader.

[0081] Figure 5 The effect of ZIF@PT on the biofilm, morphology and intracellular structure of Aeromonas veseri according to one embodiment of the present application is shown; wherein, Figure 5 (a) The inhibitory effect of ZIF-8@PT on Aeromonas vernix biofilm formation according to one embodiment of the present application; Figure 5 (b) is the removal effect of ZIF-8@PT on the established Aeromonas victoriae biofilm according to one embodiment of the present application. Figure 5 (a)- Figure 5 As shown in (b), to evaluate whether ZIF-8@PT affects biofilm formation, researchers used crystal violet staining. The results showed that adding ZIF-8@PT significantly inhibited biofilm formation when a biofilm had not yet formed. This suggests that ZIF-8@PT may affect biofilm formation by affecting bacterial growth. However, when the bacteria were cultured for 24 hours to ensure biofilm formation before adding ZIF-8@PT, they also eliminated 51.5% of the biofilm compared to the PBS-treated control group. This suggests that ZIF-8@PT not only inhibits biofilm formation by inhibiting growth, but also has a certain effect on eliminating existing biofilms.

[0082] Example 7 Bacterial morphology after ZIF-8@PT treatment

[0083] The morphology of Aeromonas veronii C4 treated with PBS, ZIF-8@PT and PT was observed using scanning electron microscopy (SEM, Zeiss Sigma 300, Germany) and transmission electron microscopy (TEM, FEI Talos F200X, USA). 6 Aeromonas veronii C4 was incubated with PBS, PT (32 μg / mL), and ZIF-8@PT (8 μg / mL). After treatment at 30°C for 12 hours, the bacteria were harvested and fixed with 2.5% glutaraldehyde. Gradient dehydration and embedding were performed to obtain 70 μm thin sections, which were then observed under a transmission electron microscope. Additionally, the gradient-dehydrated bacteria were soaked on silicon wafers and imaged using a scanning electron microscope.

[0084] Figure 5 The effect of ZIF@PT on the biofilm, morphology and intracellular structure of Aeromonas veseri according to one embodiment of the present application is shown; wherein, Figure 5 (c) is a scanning electron microscopy (SEM) image of Aeromonas vernix treated with ZIF-8 and PBS for 24 hours according to one embodiment of the present application; Figure 5 (d) is a transmission electron microscopy (TEM) image of Aeromonas vernix treated with ZIF-8, PT, and ZIF-8@PT for 24 hours according to one embodiment of the present application. Figure 5 (c)- Figure 5As shown in (d), both the morphology and intracellular structure of Aeromonas vermiformis treated with PT or ZIF-8@PT changed. In some embodiments, antimicrobial agents containing the ZIF-8@PT complex can inhibit the formation of A. vermiformis biofilms and eliminate existing biofilms. Furthermore, in some embodiments, after treatment with a fungicide containing the ZIF-8@PT complex, collapse, shrinkage, and some ruptures can be clearly observed on the surface of the A. vermiformis bacteria.

[0085] Example 8: ZIF-8@PT inhibits abdominal infection without producing toxicity

[0086] The applicant verified that ZIF-8@PT has the ability to treat abdominal infections and evaluated the prospects of ZIF-8@PT in treating abdominal infections caused by multidrug-resistant bacteria.

[0087] Figure 6 (a) is a treatment experimental design for Aeromonas welchii infection according to one embodiment of the present application. Figure 6 As shown in (a), in this embodiment, the applicant infected mice with Aeromonas veronii C4 and injected 4×10 6 CFU Aeromonas vermiformis were administered to mice treated with ZIF-8@PT. Mice treated with PBS, kanamycin (Kan), and free pyrithione (PT) served as controls.

[0088] Figure 6 (b) is an agar plate showing bacterial cultures in major organ tissues of mice treated with PBS, pyrithione (PT), ZIF-8@PT, and kanamycin (Kan), according to an embodiment of the present application. Applicants further evaluated bacterial counts in organs of infected mice treated with PBS, pyrithione (PT), kanamycin, and ZIF-8@PT. Figure 6 (c) shows the bacterial counts in the hearts of mice treated with PBS, PT, ZIF-8@PT, and kanamycin (Kan) according to an embodiment of the present application; Figure 6 (d) shows the bacterial counts in the liver of mice treated with PBS, PT, ZIF-8@PT, and kanamycin (Kan) according to an embodiment of the present application; Figure 6 (e) shows the bacterial counts in the spleen of mice treated with PBS, PT, ZIF-8@PT, and kanamycin (Kan) according to an embodiment of the present application; Figure 6 (f) shows the bacterial counts in the lungs of mice treated with PBS, PT, ZIF-8@PT, and kanamycin (Kan) according to an embodiment of the present application; Figure 6(g) is the bacterial count in the kidneys of mice treated with PBS, PT, ZIF-8@PT and kanamycin (Kan) according to an embodiment of the present application. Figure 6 (b)- Figure 6 As shown in (g), Aeromonas wilkeri proliferated significantly in the main tissues of the heart, liver, spleen, lung, and kidney, with an average bacterial count of approximately 1×10 6 Compared with free PT, ZIF-8@PT and kanamycin significantly reduced the bacterial counts in the heart, spleen, lungs, and kidneys of infected mice within 16 h, indicating that ZIF-8@PT is superior to clinical antibiotics in treating infections caused by multidrug-resistant bacteria.

[0089] Figure 6 (h) is the survival rate of infected mice after treatment with PBS, PT, ZIF@PT and kanamycin (Kan) according to an embodiment of the present application. Figure 6 As shown in (h), mice in the PBS and PT groups died within 12 hours after infection with Aeromonas vernix. Meanwhile, the survival rate of mice infected with kanamycin (Kan) was 60% at 14 hours post-infection, while the survival rate of mice infected with ZIF-8@PT was 100% at 14 hours post-infection, indicating that ZIF-8@PT can significantly improve the survival rate of infected mice compared with free PT and kanamycin.

[0090] In addition, the applicant evaluated the efficacy of ZIF-8@PT in treating infection by measuring inflammatory cytokines. Figure 6 (i) is the level of IL-4 in the serum of the control group and infected mice after treatment according to one embodiment of the present application; Figure 6 (j) is the level of IL-6 in the serum of the control group and infected mice after treatment according to one embodiment of the present application; and Figure 6 (k) is the level of IL-1β in the serum of the control group and infected mice after treatment according to one embodiment of the present application. Figure 6 (i)- Figure 6 As shown in (k), ZIF-8@PT and kanamycin (Kan) reduced the levels of IL-1β, IL-4, and IL-6, restoring them to levels comparable to the normal physiological range.

[0091] Therefore, due to its specific dual-selective mechanism, ZIF-8@PT is suitable for clinical antibiotic treatment of multidrug-resistant bacteria caused by abdominal infections and exhibits good therapeutic ability.

[0092] Figure 7This is a test of the drug resistance of Aeromonas vermiformis to ZIF-8@PT according to one embodiment of the present invention. It is noteworthy that during the extensive experimental process lasting more than a month, the inventors did not find any drug-resistant bacteria among the pathogens containing the nanocomplex of the present invention.

[0093] The above embodiments are only used to illustrate the present application and are not intended to limit the present application. Ordinary technicians in the relevant technical field can make various changes and modifications without departing from the scope of the present application. Therefore, all equivalent technical solutions should also fall within the scope disclosed in the present application.

Claims

1. A nanomolecular complex comprising: Zeolitic imidazolate framework ZIF-8; as well as Pyrithione (PT); Wherein, the pyrithione (PT) is supported on the zeolite imidazolate framework ZIF-8; Among them, the molar mass ratio of ZIF-8 to PT is (8-15):

10.

2. The nanomolecular complex according to claim 1, wherein The mass ratio of ZIF-8 to PT is (0.5-3.5):

1.

3. The nanomolecular complex according to claim 1, wherein The zeolite imidazolate framework ZIF-8 includes zinc sulfate (ZnSO4) and 2-methylimidazole, which are formed in an aqueous solution; wherein Zn 2+ , 2-methylimidazole and water has a molar ratio of 1: (65-75): (1000-1500). The nanomolecular complex according to claim 1 , wherein the size is (180-210) nm and the zeta potential is (15-40) mV. The nanomolecular complex according to claim 4 , wherein the size is 193.80±2.68 nm and the zeta potential is -27.80±4.98 mV.

6. A nano antibacterial agent comprising the nano molecular complex according to any one of claims 1 to 5.

7. Use of the nanomolecular complex according to any one of claims 1 to 5 in the preparation of nano antibacterial agents for preventing and / or treating diseases caused by pathogens.

8. The use according to claim 7, comprising administering an effective dose of the nano antibacterial agent to the pathogen, wherein the effective administration dose is a concentration of the nano complex of not less than 3 μg / ml.

9. The use according to claim 8, comprising administering an effective dose of nano antimicrobial agent to pathogens, wherein: The effective dose for inhibiting the growth of pathogens is a concentration of the nanocomplex of not less than 3.5 μg / ml; the effective dose for killing pathogens is a concentration of the nanocomplex of not less than 7 μg / ml.

10. The use according to claim 9, wherein: The effective dose for inhibiting the growth of pathogens is a concentration of the nanocomplex of not less than 4 μg / ml; the effective dose for killing pathogens is a concentration of the nanocomplex of not less than 8 μg / ml.

11. A method for killing or inhibiting the growth of pathogens in vitro, comprising administering an effective dose of the nanocomplex according to any one of claims 1 to 5 or the nano antibacterial agent according to claim 6 to the pathogens.

12. Use of the nanocomposite according to any one of claims 1 to 5 or the nano antibacterial agent according to claim 6 in the preparation of a medicament for preventing and / or treating pathogen infection in fish or mammals.

13. The use according to claim 12, wherein the infection is an abdominal infection.

Images