A silver-based porous organic framework material and its preparation method and application

Abstract

The invention discloses a silver-based porous organic framework material and a preparation method and application thereof. First, 4,4′-diformyldibenzo-18-crown-6 is prepared from dibenzo-18-crown-6, trifluoroacetic acid and hexamethylenetetramine; then, CEP-POP is obtained by reflux reaction of redistilled pyrrole, 4,4′-diformyldibenzo-18-crown-6 and FeCl2·4H2O; finally, CEP-POP is mixed with SNP, and a silver nitrate solution is added for mixed reaction to obtain CEP-POP-AgNNP. In the invention, silver nitroprusside and CEP-POP are combined to achieve synergistic antibacterial; and the silver-based material is not easy to fall off from the carrier, will not self-aggregate, has good biocompatibility, significantly reduces the toxicity of the silver-based nanoparticle antibacterial agent, and has no toxic side effects.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a silver-based porous organic framework material and a preparation method and application thereof. Background Art

[0002] Bacteria are one of the main pathogenic microorganisms that cause infection. The invasion of pathogens on the human body has promoted human research on pathogens. The discovery of penicillin in 1929 opened up the way for antibiotics to treat bacterial infections. The rapid development of antibiotics in the middle of the 20th century greatly reduced the morbidity and mortality of bacterial infections and improved the quality of life and life expectancy of infected humans. However, antimicrobial resistance (AMR) has also followed, which can be mainly attributed to two points: 1) multidrug resistance caused by long-term excessive use of antimicrobial drugs by patients and 2) clinical abuse of antibiotics in the past few years. The two together have exacerbated this hazard, making the emergence of AMR the biggest threat in modern medicine in recent years. Therefore, a new strategy is urgently needed to combat bacterial infections and overcome the current situation of drug resistance. In recent years, the advent and rapid development of nanomaterials have provided possibilities and opportunities to overcome the current situation of antibiotic-resistant bacteria.

[0003] Silver-based antimicrobial agents are currently the most widely studied alternatives for eliminating drug-resistant strains. However, as a typical heavy metal, silver-based antimicrobial agents, including metallic silver, silver salts, and silver nanoparticles, encounter the problem of systemic side effects, which may lead to allergic reactions and severe cytotoxicity, especially at high doses. At the same time, the inherent self-aggregation properties of silver-based nanoparticles greatly reduce the bactericidal effect of silver-based antimicrobial agents. In order to further apply silver-based antimicrobial agents in clinical practice, new carriers are urgently needed that can not only improve biocompatibility but also avoid the severe self-aggregation of silver-based nanoparticles, so as to play a better therapeutic role.

[0004] Porous organic polymers (POPs), similar to but different from graphene materials (GMs), single-walled or multi-walled carbon nanotubes (CNTs) and metal organic frameworks, are a subclass of artificial porous materials connected by light elements through covalent bonds. The inherent structural properties similar to proteins give persistent organic pollutants good biosafety, while the adjustable chemical structure and functions make them useful in but not limited to the environment, catalysis, biosensors, energy and biomedicine. Therefore, how to construct multifunctional POPs with intrinsic antibacterial effects, which can load silver-based materials to achieve synergistic sterilization, avoid self-aggregation of silver-based materials or detachment from the carrier, and reduce the toxicity of silver nanoparticles, is a problem that needs to be solved. Summary of the invention

[0005] In view of the above-mentioned prior art, the purpose of the present invention is to provide a silver-based porous organic framework material and its preparation method and application. The present invention combines the action of silver nitroprusside with the CEP-POP crown ether-based porous organic framework to prepare a broad-spectrum antibacterial agent. The synergistic antibacterial effect of silver nitroprusside and CEP-POP is achieved, achieving a significant antibacterial effect; and the silver-based material is not easy to fall off from the carrier, will not self-aggregate, has good biocompatibility, significantly reduces the toxicity of the silver-based nanoparticle antibacterial agent, and has no toxic side effects.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The first aspect of the present invention provides a method for preparing a silver-based porous organic framework material, comprising the following steps:

[0008] (1) Preparation of 4,4′-diformyldibenzo-18-crown-6: dibenzo-18-crown-6, trifluoroacetic acid and hexamethylenetetramine were mixed, stirred and heated for reaction under a protective atmosphere, and after the reaction was completed, the mixture was cooled to obtain a yellow-brown precipitate, which was washed, filtered and dried to obtain 4,4′-diformyldibenzo-18-crown-6;

[0009] (2) Preparation of CEP-POP: Add redistilled pyrrole, 4,4′-diformyldibenzo-18-crown-6 obtained in step (1) and FeCl2·4H2O to propionic acid, reflux under a protective atmosphere, wash and dry the obtained black powder to obtain CEP-POP;

[0010] (3) Preparation of CEP-POP-AgNNP: CEP-POP and SNP obtained in step (2) are added to water to obtain a mixed solution, which is stirred at room temperature overnight. After centrifugation, the obtained precipitate is washed and freeze-dried to obtain SNP@CEP-POP. Silver nitrate solution is added to the obtained SNP@CEP-POP, and the mixture is stirred at room temperature for reaction. The solid obtained by the stirring reaction is washed and dried to obtain a porous organic framework material, namely CEP-POP-AgNNP.

[0011] Preferably, in step (1), the ratio of the added amounts of dibenzo-18-crown-6-ether, hexamethylenetetramine and trifluoroacetic acid is 10 mmol:10 mmol:7 mL.

[0012] Preferably, in step (1), the protective atmosphere is argon; the temperature of the heating reaction is 90° C., and the time of the heating reaction is 12 to 15 hours.

[0013] Preferably, in step (2), the ratio of the added amounts of redistilled pyrrole, 4,4′-diformyldibenzo-18-crown-6, FeCl2·4H2O and propionic acid is 4 mmol:4 mmol:1.2 mmol:120 mL.

[0014] Preferably, in step (2), the protective atmosphere is argon; the temperature of the reflux reaction is 140° C., and the time of the reflux reaction is 48 h.

[0015] Preferably, in step (3), the mass ratio of CEP-POP to SNP is 1:12, and the concentration of CEP-POP in the mixed solution is 2.5 mg / mL.

[0016] Preferably, in step (3), the ratio of the amount of SNP@CEP-POP to the amount of silver nitrate solution added is 65:68; the concentration of the silver nitrate solution is 1×10 -3 M; the stirring reaction time is 3h.

[0017] The second aspect of the present invention provides a silver-based porous organic framework material obtained by the above preparation method.

[0018] The third aspect of the present invention provides the use of a silver-based porous organic framework material in the preparation of drugs against Gram-negative bacteria and Gram-positive bacteria.

[0019] Preferably, the Gram-negative bacteria is Escherichia coli; and the Gram-positive bacteria is Staphylococcus aureus.

[0020] Beneficial effects of the present invention:

[0021] (1) The present invention combines silver nitroprusside with CEP-POP crown ether-based porous organic framework to prepare a silver-based porous organic framework material that can not only load silver-based materials and release silver nitroprusside nanoparticles to achieve synergistic bactericidal effects, but also prevent the silver-based materials from self-aggregating or falling off the carrier, thereby achieving a synergistic broad-spectrum antibacterial effect of porous organic polymer materials.

[0022] (2) The silver-based porous organic framework material CEP-POP-AgNNP prepared by the present invention has good biocompatibility, a hemolysis rate of less than 1% on red blood cells, no toxic side effects on the human body, and little effect on the cell viability of HEK293 cells, which significantly reduces the toxicity of silver-based nanoparticle antibacterial agents, which will promote the development of multifunctional antibacterial platforms containing silver-based nanoparticles. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The characterization spectra of CEP-POP-AgNNP, where (a) is the infrared spectra of CEP-POP-AgNNP, AgNNP, CEP-POP and SNP; (b) is the infrared spectra of CEP-POP-AgNNP 13C NMR solid carbon spectrum; (c) XRD patterns of CEP-POP-AgNNP and CEP-POP; (d) TGA patterns of CEP-POP-AgNNP and CEP-POP; (e) low-temperature N2 absorption isotherms of CEP-POP and CEP-AgPOP at 77K; (f) pore size distribution curves of CEP-POP and CEP-POP-AgNNP;

[0024] Figure 2 The electron microscope images and element mapping of CEP-POP-AgNNP, where (a) is the scanning electron microscope of CEP-POP-AgNNP at 5μm; (b) is the scanning electron microscope of CEP-POP-AgNNP at 100nm; (c) is the transmission electron microscope of CEP-POP-AgNNP at 500nm; (d) is the transmission electron microscope of CEP-POP-AgNNP at 200nm; (e) is the transmission electron microscope of CEP-POP-AgNNP at 200nm; (f) is the HRTEM of CEP-POP-AgNNP at a scale of 50nm; (g) is the HRTE of CEP-POP-AgNNP at a scale of 5nm M; (h) is HRTEM of CEP-POP-AgNNP at a scale of 5 nm; (i) is element mapping of CEP-POP-AgNNP at a scale of 50 nm; (j) is C element mapping of CEP-POP-AgNNP at a scale of 50 nm; (k) is N element mapping of CEP-POP-AgNNP at a scale of 50 nm; (l) is O element mapping of CEP-POP-AgNNP at a scale of 50 nm; (m) is Fe element mapping of CEP-POP-AgNNP at a scale of 50 nm; (n) is Ag element mapping of CEP-POP-AgNNP at a scale of 50 nm;

[0025] Figure 3 is the Zeta potential distribution diagram of CEP-POP-AgNNP, AgNNP, and CEP-POP;

[0026] Figure 4 is the EDX image of CEP-POP-AgNNP;

[0027] Figure 5 is the antibacterial activity of CEP-POP, where (a) is the colony photo formed by plate count treatment of CEP-POP and E. coli; (b) is the survival rate of Staphylococcus aureus and E. coli after CEP-POP treatment;

[0028] Figure 6is an in vitro bacterial growth inhibition measurement; where (a) is based on the plate count method (concentration: CEP-POP-AgNNP = 0, 1, 3, 5 μg mL -1 , AgNNPs = 0, 0.4, 1 and 2 μg mL -1 , CEP-POP=0, 0.6, 2.0 and 3.0 μg mL -1 ), photos of colonies formed by CEP-POP-AgNNPs, AgNNPs, and CEP-POP after Staphylococcus aureus;

[0029] (b) is the survival rate of Staphylococcus aureus after CEP-POP treatment; (c) is the survival rate of Staphylococcus aureus after AgNNPs treatment; (d) is the survival rate of Staphylococcus aureus after CEP-POP-AgNNP treatment; (e) is the survival rate of Staphylococcus aureus based on the plate count method (concentration: CEP-POP-AgNNP = 0, 1, 3, 5 μg mL -1 , AgNNPs = 0, 0.4, 1 and 2 μg mL -1 , CEP-POP = 0, 0.6, 2 and 3 μg mL -1 ) Photos of colonies formed after E. coli was treated with CEP-POP-AgNNPs, AgNNPs and CEP-POP; (f) is the viability of E. coli after CEP-POP treatment; (g) is the survival rate of E. coli after AgNNPs treatment; (h) is the survival rate of E. coli after CEP-POP-AgNNP treatment;

[0030] Figure 7 are the fluorescence images and electron microscopy images of Staphylococcus aureus and Escherichia coli after treatment in each group, where (a) is the fluorescence image of Staphylococcus aureus and Escherichia coli co-incubated with PBS, CEP-POP, AgNNPs, and CEP-POP-AgNNPs, obtained by PI and SYTO-9 co-staining test (scale bar = 200 μm), I-IV) Staphylococcus aureus obtained after treatment with PBS, CEP-P-POP, AgNNPs, and CEP-POP-AgNNPs; V-VIII) PBS, CEP-POP, AgNNPs, E. coli obtained after treatment with CEP-POP-AgNNPs; (b) TEM images of E. coli incubated with Staphylococcus aureus and PBS, CEP-POP, AgNNPs, and AgNP-PEP-PGNNPs (scale bar = 2 μm), I-IV) Staphylococcus aureus obtained after treatment with PBS, CEP-P-POP, AgNNPs, and CEP-POP-AgNNPs; V-VIII) E. coli obtained after treatment with PBS, CEP-POP, AgNNPs, and CEP-POP-AgNNPs;

[0031] Figure 8 It is the biocompatibility verification of CEP-POP-AgNNP, where (a) is the comparison of hemolysis test of CEP-POP at different concentrations; (b) is the hemolysis test of AgNNPs; (c) is the hemolysis test of CEP-POP-AgNNP; (d) is the comparison of hemolysis test of AEP-POP; (e) is the viability (%) of HEK293 cells treated with different concentrations of CEP-POP; (f) is the comparison of the viability (%) of HEK293 cells treated with CEP-POP-AgNNP, AgNNPs and CEP-POP;

[0032] Fig. 9 It is the verification of the in vivo antibacterial effect of CEP-POP, AgNNPs, and CEP-POP-AgNNPs, where (a) is the representative wound photos of the wounds infected with Staphylococcus aureus on the back of BALB / c mice at 6, 4, 6, 8, and 10 days after CEP-POP, AgNNPs, and CEP-POP; (b) is the change of wound healing area during treatment; (c) is the change of mouse body weight during treatment, n=5; error bars represent standard deviation;

[0033] Fig.10 The results of H&E and Masson trichrome staining of the tissues in each wound group on the 10th day. The red boxes represent subcutaneous hemorrhage; the green boxes represent blood vessels; the yellow boxes represent hair follicles. The scale bar is 500 μm.

[0034] Fig.11 are representative H&E staining images of the main organs (including heart, liver, spleen, lung, and kidney) collected from each group after 10 days of treatment;

[0035] Fig.12 These are the hematological examination results of different groups (day 10), where (a) is the RBC examination result, (b) is the HGB examination result, (c) is the WBC examination result, (d) is the MCV examination result, (e) is the MCHC examination result, (f) is the HCT examination result, (g) is the MCH examination result, and (h) is the PLT examination result. DETAILED DESCRIPTION

[0036] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0037] As described in the background technology section, compared with traditional inorganic metal materials, porous organic polymers have good biocompatibility and low toxicity, which makes them safer for use in organisms. So far, many porous organic polymers have been successfully prepared for antibacterial applications. Therefore, if silver-based materials are loaded in porous organic polymers, the bactericidal effect can be improved; however, the characteristics of Ag-based antibacterial agents themselves and their severe cytotoxicity greatly hinder their clinical application.

[0038] Based on this, the purpose of the present invention is to provide a silver-based porous organic framework material and a preparation method and application thereof. The porous organic polymer CEP-POP-AgNNP prepared by the present invention is based on crown ethers and porphyrins as structural units, and has good biocompatibility that inorganic metal compounds do not have. The present invention is the first to confine ultrafine silver nitroprusside nanoparticles (AgNNPs) in the void space of the crown ether-based POP carrier, which not only significantly and effectively reduces the toxicity of the nanoparticles, but also avoids the direct interaction between the silver-based antibacterial agent and the external environment, thereby greatly improving its stability. The synergistic effect of CEP-POP and AgNNPs makes CEP-POP-AgNNP have a very low dose (5μg mL -1 ) can have significant bactericidal ability against Gram-negative (G-) and Gram-positive (G+) strains. Under normal circumstances, at the concentration of the present invention, silver nanoparticles will kill cells, but the present invention can make silver nanoparticles obtain the same bactericidal effect as silver nitroprusside while maintaining high cell activity. At the same time, CEP-POP-AgNNP has a negligible hemolysis rate and negligible normal cell damage, which makes it have great potential in biomedicine. Therefore, the present invention paves the way for the preparation of promising combined antibacterial drugs for antibacterial-related biomedical applications.

[0039] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0040] Description: The redistilled pyrrole used in the present invention is prepared by the following method: prepare a 50mL round-bottom flask with a magnetic stirrer, add 3mL pyrrole thereto, then connect a 25mL constant pressure separatory funnel to the mouth of the round-bottom flask, and close the knob of the constant pressure separatory funnel so that it does not circulate with the round-bottom flask below. A spherical condenser is installed at the upper end of the constant pressure separatory funnel, and a single-pass interface is installed at the upper end of the spherical condenser. The above-mentioned device is placed in a 90°C water bath, the vacuum pump's exhaust rubber hose interface is connected to the single-pass interface, the vacuum pump is turned on and stirring is started at the same time. The pyrrole redistillation process is completed when there is no significant liquid residue in the round-bottom flask below the device, the pyrrole obtained by redistillation is condensed and stored in a constant pressure separatory funnel, and then an appropriate amount is taken and put into the subsequent reaction system.

[0041] The English name of dibenzo-18-crown-6 used in the present invention is Dibenzo-18-Crown-6, which can also be translated into dibenzo-18-crown-6-ether or dibenzo-18-crown ether-6. 4,4′-diformyl dibenzo-18-crown-6 is the same as above.

[0042] The test materials used in the examples of the present invention are all conventional test materials in the art and can be purchased through commercial channels.

[0043] Example 1

[0044] (1) Synthesis of 4,4′-diformyldibenzo-18-crown-6:

[0045] Dibenzo-18-crown-6 (10 mmol), CF3COOH (7 ml) and hexamethyltetramine (urotropine) (10 mmol) were added to a 50 ml three-necked flask with rubber stoppers on both sides, and the air in the reaction vessel was replaced with argon. The mixture was stirred at 90 ° C for 12 hours, and the reaction process was detected by thin layer chromatography. After the reaction, the mixture was slowly poured into cold water at room temperature and vigorously stirred until a yellow-brown solid precipitate was generated. The precipitated solid was washed and filtered twice with distilled water. The product was recrystallized with anhydrous ethanol as solvent at a recrystallization temperature of -20 ° C. After filtering through a Buchner funnel, it was vacuum dried at room temperature at a drying temperature of 40 ° C to obtain a pure product. 4. The synthetic route of 4′-diformyldibenzo-18-crown-6 is:

[0046]

[0047] (2) Preparation of CEP-POP:

[0048] In a 250mL three-necked flask, freshly distilled pyrrole (redistilled pyrrole) (16mmol) and 4,4′-diformyldibenzo-18-crown-6 (4mmol) were added to 120mL of propionic acid. Then, FeCl2·4H2O (4.8mmol) was added to the above mixture, the air in the reaction vessel was replaced with argon, and the mixture was refluxed at 140°C in an argon atmosphere for 2 days to obtain a black powder, which was washed twice with ethanol and tetrahydrofuran respectively. Finally, it was dried in a vacuum oven at 60°C, and then Soxhlet extraction was performed with dichloromethane and methanol for 24h at 80°C, and then strictly washed with distilled water and dried in a vacuum oven at 80°C to obtain a black solid powder CEP-POP. The synthetic route of CEP-POP is:

[0049]

[0050] (3) Preparation of CEP-POP-AgNNPs

[0051] 5 mg CEP-POP and 60 mg SNP were added to 2 ml water and stirred overnight at room temperature. The precipitate was centrifuged, washed twice with distilled water, and then freeze-dried to obtain SNP@CEP-POP. 40 mL silver nitrate solution (1×10 -3 M) and 65 mg of SNP@CEP-POP were added into a 250 ml round-bottom bottle, stirred vigorously at room temperature for 3 h at a stirring speed of 450 rpm, centrifuged at 10,000 rpm for 40 min with distilled water, centrifuged twice, and placed in a vacuum drying tank overnight at a drying temperature of 80 ° C to obtain CEP-POP-AgNNP.

[0052] Example 2

[0053] (1) Synthesis of 4,4′-diformyldibenzo-18-crown-6:

[0054] Dibenzo-18-crown-6 (10 mmol), CF3COOH (7 ml) and hexamethyltetramine (10 mmol) were added to a 50 ml three-necked flask with rubber stoppers on both sides, and the air in the reaction vessel was replaced with argon. The mixture was stirred at 90 ° C for 15 hours, and the reaction process was detected by thin layer chromatography. After the reaction, the mixture was slowly poured into cold water under vigorous stirring at room temperature until a yellow-brown solid precipitate was generated. The precipitated solid was washed and filtered 3 times with distilled water. The product was recrystallized with anhydrous ethanol as solvent at a recrystallization temperature of -20 ° C. After filtering through a Buchner funnel, it was vacuum dried at room temperature at a temperature of 40 ° C to obtain a pure product.

[0055] (2) Preparation of CEP-POP

[0056] In a 250mL three-necked flask. Freshly distilled pyrrole (16mmol) and 4,4′-diformyldibenzo-18-crown-6 (4mmol) were added to 120mL of propionic acid. Then, ferrous chloride tetrahydrate (4.8mmol) was added to the above mixture, the air in the reaction vessel was replaced with argon, and the mixture was refluxed at 140°C in an argon atmosphere for 2 days. A black powder was obtained, which was washed three times with ethanol and tetrahydrofuran. Finally, it was dried in a vacuum oven at 60°C, and then Soxhlet extraction was performed with dichloromethane and methanol for 24h in sequence at an extraction temperature of 80°C, and then strictly washed with distilled water and dried in a vacuum oven at 80°C to obtain a black solid powder CEP-POP.

[0057] (3) Preparation of CEP-POP-AgNNPs

[0058] 5 mg CEP-POP and 60 mg SNP were added to 2 ml water and stirred overnight at room temperature. The precipitate was centrifuged, washed with distilled water three times, and then freeze-dried to obtain SNP@CEP-POP. 40 mL silver nitrate solution (1×10 -3 M) and 65 mg SNP@CEP-POP were added into a 250 ml round-bottom bottle, stirred vigorously at room temperature for 3 h at a stirring speed of 450 rpm, centrifuged at 10,000 rpm for 40 min with distilled water, centrifuged twice, and placed in a vacuum drying tank overnight at a drying temperature of 80 ° C to obtain CEP-POP-AgNNP.

[0059] The CEP-POP-AgNNP prepared in Example 1 was characterized:

[0060] (1) Figure 1 As shown in (a), the presence of crown ether and metalloporphyrin units in CEP-POP was initially confirmed by Fourier transform infrared spectroscopy (FT-IR). -1 , 1685cm -1 ) and pyrrole (3413cm -1 , 1301cm -1 In addition to the characteristic vibrations of pyrrole (NH and CN stretching vibrations), the wavelength of 1008 cm -1 The new absorption band centered at 1685 cm was identified as the Fe-N stretching vibration unique to the cross-linked unit of iron porphyrin. -1) The stretching vibration of CEP-POP again supports the successful synthesis of CEP-POP. In addition, the FITR of CEP-POP-AgNNPs integrates the characteristics of POP and AgNNPs simultaneously. It can be clearly seen that the 2143 cm-1 in SNPs is replaced by the Fe-Ag connection due to the replacement of the Fe-Na connection. -1 The weak absorption of -CN bond at AgNNPs and CEP-POP-AgNNPs shifted to 2177 cm- -1 This can be attributed to the electronegativity between cations (Ag + The electronegativity of Na is 1.93 eV. + electronegativity is 0.93eV) and ion size (Ag + The electronegativity of Na+ is 0.126nm, and the electronegativity of Na+ is 0.102nm). The electron induction effect increases with the increase of electronegativity and ion size, causing the absorption peak to shift to higher wavenumbers.

[0061] (2) Through Figure 1 (b) Solid 13 C -NMR further confirmed the skeleton structure of CEP-POP-AgNNP. Obvious carbon signals (115, 128 and 147.5 ppm, belonging to -C=C-, Ar -C＝C- The signal at 124.4 ppm belongs to -C≡N in AgNNPs. The above evidences support the successful synthesis of CEP-POP-AgNNPs.

[0062] (3) In order to check the crystallinity of the material, XRD detection was performed on the CEP-POP and CEP-POP-AgNNP prepared in Example 1. Figure 1 (c). The XRD of CEP-POP prepared in Example 1 shows no characteristic peaks, revealing an amorphous skeleton structure. The diffraction peaks of CEP-POP-AgNNP show the crystalline nature of AgNNPs, and typical peaks are observed at 2θ=13.8, 19.2, 21.6, 25.3, 27.78 and 29.78. These diffraction peaks point to the (010), (110), (210), (-112), and (021) crystal planes of AgNNPs. The unit cell parameters are a=7.5218, b=6.4087, c=11.0134, α=90.00000°β=102.06°, γ=90.00000°, indicating the crystalline nature of the monoclinic unit cell of AgNNPs.

[0063] (4) Figure 1 (d) Thermogravimetric analysis shows the thermal stability of CEP-POP-AgNNPs. Thermogravimetric analysis (TGA) thermal stability test shows that CEP-POP remains stable at high temperature under N2 conditions and does not decompose until 140°C. In contrast, the weight of CEP-POP-AgNNPs decreases slightly at 110°C due to the loading of AgNNPs. The difference in weight loss between CEP-POP and CEP-POP-AgNNPs is attributed to the easily degradable nature of cyanide in SNPs. Under low temperature conditions (110-250°C), the mass loss is 14%. It is mainly due to the evolution of weak binding of non-bridging NO ligands to axial CN groups. The loss above 250°C comes from the other two types of CN groups in AgNNPs. It shows that CEP-POP does not show obvious mass loss and has good thermal stability.

[0064] (5) Figure 1 (f) The porosity characteristics of the synthesized material CEP-POP-AgNNP were determined by low-temperature nitrogen adsorption. CEP-POP-AgNNP showed a typical curve that conformed to the characteristics of the IV type adsorption isotherm, which confirmed the existence of a large number of mesopores and macropores. Pore size analysis ( Figure 1f) shows that the pores are mainly distributed within 1.96-12.72 nm, with a specific surface area of ​​13.7 m 2 / g.

[0065] (6) Figure 2 (a)- Figure 2 The scanning electron microscopy (SEM) result of (b) shows the three-dimensional (3D) interconnected structure of CEP-POP-AgNNPs, which is composed of rod-like particles.

[0066] (7) Figure 2 (c)- Figure 2 The transmission electron microscopy (TEM) in (f) clearly detected the clear mesopores and irregularly distributed macropores on the porous skeleton of CEP-POP-AgNNP. Figure 2 (h) HRTEM can further verify the crystal structure of AgNNPs in CEP-POP-AgNNP, whose lattice spacing is 0.241nm, corresponding to the (210) crystal plane of AgNNPs. Figure 2 (i)- Figure 2 Elemental mapping of (n) confirmed the successful doping of AgNNPs and the uniform distribution of elements.

[0067] (8) Figure 3 Surface charge of the synthesized material CEP-POP-AgNNP collected by zeta potential. There is Fe in CEP-POP 3+ ions, CEP-POP exhibited a positive zeta potential. In contrast, pure AgNNPs showed a negative zeta potential. However, the zeta potential of CEP-POP-AgNNPs was significantly reduced compared with that of CEP-POP (the zeta potential of CEP-POP was 36.67±0.75mV and the zeta potential of CEP-POP-AgNNPs was 21.13±0.56mV), indicating that AgNNPs were loaded into the POP skeleton.

[0068] (9) Figure 4 TEM-EDS again confirmed the successful doping of AgNNPs in the porous carbon framework, with the mass ratios of uniformly distributed C, N, O, Fe and Ag being 53.3wt%, 15.2wt%, 6.18wt%, 19.5wt% and 5.81wt%, respectively.

[0069] Test Example 1: In vitro bacterial growth inhibition test

[0070] In order to comprehensively evaluate the in vitro antibacterial effect of the synthesized CEP-POP-AgNNPs prepared in Example 1 against Staphylococcus aureus and Escherichia coli, the co-culture and plate count methods were used to test the antibacterial effect of the polymer itself.

[0071] (1) Take 100uL of second generation Staphylococcus aureus (10 8 CFU mL -1 ) or Escherichia coli (10 8 CFU mL -1 ) with 100 uL PBS (pH = 7.4) and concentrations of 0.6, 2, 3, 5, 10, and 20 μg mL -1 The cells were incubated with CEP-POP in a 37°C incubator for 12 h, and PBS was used as a blank control group. Figure 5 As shown in (a), visible microbial growth was found in the PBS group (blank control group). In contrast, CEP-POP showed a dose-dependent antibacterial effect, with the highest concentration of CEP-POP (20 μg mL -1 ) had the greatest inhibitory activity against Staphylococcus aureus (59.5±6.02%) and Escherichia coli (39.7±3.51%). Figure 5 As shown in (b), at low concentrations (0-3.0 μg mL -1 ), CEP-POP had only a slight effect on the activity of Staphylococcus aureus and Escherichia coli. This result indicates that the crown ether-based porous framework has an antibacterial effect and can be used as a material-based antibacterial agent.

[0072] (2) Take 100 μL (10 5 CFU mL -1 ) and 900 μL of different treatment groups: CEP-POP-AgNNP (5, 3, and 1 μg mL -1 ) group, AgNNPs (2, 1.4 and 0.4 μg mL -1 ) group, CEP-POP (3, 1.6 and 0.6 μg mL -1 ) group and PBS (pH = 7.4) control group, inoculated into LB bacterial incubation tubes, and incubated in a constant temperature incubator (37°C) for 12 hours. Take the bacterial solution (80μL) after different sample treatments and evenly distribute it on the LB plate, then incubate it upside down in a constant temperature incubator at 37°C for 16 hours, and then count the number of colonies growing on the culture plate. All experiments were repeated 3 times. Figure 6 It can be seen that compared with the CEP-POP group, CEP-POP-AgNNP (1, 3 and 5 μg mL -1 ) group and AgNNPs (0.4, 1 and 2 μg mL -1 ) group had a more significant antibacterial effect. -1 The survival rates of bacteria treated with CEP-POP (3 μg mL-1 The survival rates of CEP-POP-AgNNPs (5 μg mL -1 The inhibitory activity of the AgNNPs group against Staphylococcus aureus and Escherichia coli reached 97.7±0.5% and 90.7±0.5%, respectively. The above experimental results show that loading AgNNPs into CEP-POP can amplify the synergistic antibacterial performance and effectively improve the antibacterial effect.

[0073] Test Example 2: Bacterial Live / Dead Staining

[0074] The results of the plate count method were further confirmed by live / dead staining experiments, using green fluorescent nucleic acid dye (SYTO-9) and red fluorescent propidium iodide (PI) nucleic acid dye to treat different groups of bacteria: 100 μL of Escherichia coli or Staphylococcus aureus (108 CFU mL -1 ), mix 900 μL PBS (pH=7.4), 900 μL CEP-POP-AgNNP (5 μg mL -1 )、900μL AgNNPs (2μg mL -1 )、900μL CEP-POP(3μg mL -1 ) were mixed with the bacterial solution and cultured in a constant temperature incubator at 37°C for 12 h. The PBS (pH = 7.4) group was used as a negative control. 200 μL of each cultured bacterial tube was taken and 20 μL of SYTO-9 (1.0 × 10 -3 M) and 20 μL PI (1.5×10 -3 M) was treated at 37°C in the dark for 15 minutes. SYTO-9 is a green fluorescent nucleic acid dye that can penetrate and bind to nucleic acids of live and dead bacteria, while red fluorescent PI can only penetrate bacteria with damaged cell membranes. SYTO-9 (green fluorescent nucleic acid stain SYTO-9) and PI (propidium iodide) are used to distinguish between live and dead microbial cells. SYTO-9 marks bacterial cells green because it penetrates all bacterial membranes (intact and damaged), while PI can only penetrate damaged cell membranes, marking cells red while reducing the green color caused by SYTO-9 staining. After staining, the sample was treated with PBS centrifugation (5min, 5000rpm) to remove excess SYTO-9 and PI. The bacteria were then resuspended in 50μL PBS and placed on the surface of the slide. Fluorescent photos of the staining were obtained using a confocal microscope (Leica.TCS SP8.GER), marking dead bacteria with red fluorescence and live bacteria with green fluorescence. As Figure 7As shown in (a), except for the PBS group, different degrees of cell death were detected in other groups after treatment, and red fluorescence was visible. Compared with CEP-POP, bacteria treated with CEP-POP-AgNNPs and AgNNPs showed stronger red fluorescence. It is worth noting that after treatment with CEP-POP-AgNNPs, almost all bacteria were labeled red, which is consistent with the results of co-culture.

[0075] Test Example 3: Transmission Electron Microscope Scanning of Bacteria

[0076] To further elucidate the basic antibacterial mechanism of CEP-POP-AgNNP, the ultrastructural damage and organizational disorder of Staphylococcus aureus and Escherichia coli were studied by comparing the transmission electron microscopy images of bacteria before and after incubation between different groups. 8 CFU mL -1 ) were compared with 5 μg mL -1 CEP-POP-AgNNP, 3 μg mL -1 AgNNPs, 2 μg mL -1 CEP-POP and PBS (pH = 7.4) were incubated for 12 hours, recorded as CEP-POP-AgNNP group, AgNNPs group, CEP-POP group and control group. The bacteria after different treatments were collected and centrifuged at 5000 rpm for 3 minutes. The concentrated bacterial solution was fixed with 2.5% glutaraldehyde for 2 hours and 1% osmium tetroxide aqueous solution for 2 hours at room temperature. Then, the samples were washed 3 times with PBS, treated in an ascending ethanol series (volume fractions of 30, 50, 70, 80, 90 and 100%) for 10 minutes, and treated with embedding medium (volume ratio of acetone: Epon 812 = 1:1) and pure Epon 812 in sequence for 12 hours. The samples were then embedded at 60°C for 48 hours. Sections (60-80 nm) were prepared and stained with uranyl acetate using a microtome equipped with a diamond knife). Finally, the thin slices containing cells were placed on copper grids for transmission electron microscopy observation. As Figure 7 As shown in (b), the cell walls of Staphylococcus aureus and Escherichia coli in the two control groups survived, and the cell walls were smooth and intact. In contrast, cell wall ruptures of varying degrees were found in the other groups. For example, the bacteria under CEP-POP treatment only showed slight cell wall ruptures and structural deformations. After AgNNPs treatment, the bacterial morphology changed significantly, the bacterial membrane was destroyed, and the cytoplasm flowed out. In the CEP-POP-AgNNP group, both bacteria were severely damaged, and their cell membranes completely collapsed due to the leakage of intracellular components, eventually leading to bacterial death.

[0077] Test Example 4: Hemolysis Test

[0078] Fresh blood was collected from 8-week-old BALB / c female mice (purchased from Jinan Jerecon Biotechnology Co., Ltd.) and red blood cells were collected by centrifugation at 1500 rpm for 20 min and washed three times with PBS (pH = 7.4). Red blood cells (4% w / w) were mixed with PBS solutions of CEP-POP, AgNNPs, and CEP-POP-AgNNPs (all in the concentration range of 3-5 μg mL -1 ) were mixed in a ratio of 1:9 (v / v) and incubated in a shaker at 37°C for 3h. Red blood cells (4% w / w) were mixed with distilled water and PBS (pH = 7.4) and incubated in a shaker at 37°C for 3h. The distilled water solution was used as a positive control (+) and PBS (pH = 7.4) was used as a negative control (-). Subsequently, the mixture was centrifuged at 12000 rpm for 20 min. The hemolytic effect of CEP-POP-AgNNP was detected by measuring the absorbance of each supernatant at 540 nm by UV-visible spectroscopy. Figure 8 (a)- Figure 8 (d) is the hemolysis rate of CEP-POP, AgNNPs, and CEP-POP-AgNNP on red blood cells at different concentrations. It can be seen from the figure that the concentration of CEP-POP-AgNNP is 5 μg mL -1 , the hemolysis rate is still maintained below 1%, which shows that the material has good biocompatibility.

[0079] Test Example 5: Cytotoxicity Test

[0080] Cytotoxicity is also an important indicator for evaluating the biocompatibility of materials. Human embryonic kidney cells (HEK293 cell line, from the School of Pharmacy of Weifang Medical College) were used for testing.

[0081] HEK293 cells were plated at 8 × 10 cells per well in a 96-well plate. 3 Each well was inoculated with 100 μL of cell suspension, and 100 μL of PBS was added to the surrounding duplicate wells for liquid sealing to prevent excessive evaporation. After 24 hours of incubation, different concentrations of CEP-POP (1-5 μg mL -1 )、AgNNPs(1~5μg mL -1 )、CEP-POP-AgNNP(1~5μgmL -1 ) and DMEM basal medium (control group). After 24 h, the supernatant was discarded and 10 μL MTT (5 μg mL -1 ) The same volume of PBS (pH = 7.4) solution was added to each well. After 4 hours, the supernatant was aspirated and 100 μL of DMSO was added to each well to dissolve the MTT-formamide crystals. After 5 minutes, the absorbance was measured at a wavelength of 490 nm in an ELISA reader. Figure 8 (e)- Figure 8 (f) It can be seen that with the increase of CEP-POP-AgNNP concentration, the survival rate of HEK293 cells slowly decreased after 24 h. -1 When the cell culture medium was added with 5% iodine, the survival rate of HEK293 cells was still maintained above 80%, which once again demonstrated that CEP-POP-AgNNP has good biocompatibility.

[0082] Test Example 5: Wound healing test

[0083] To further evaluate the potential for in vivo applications, a skin wound model of Staphylococcus aureus infection was established in 6-week-old female BALB / C mice. The dorsal hair of each mouse was shaved, a 6 mm wound was formed by perforation, and then 20 μL of Staphylococcus aureus (1×10 6 CFU mL -1 ) were infected for 48 h. -1 )、CEP-POP (100 μL, 5 μg mL -1 ) and CEP-POP-AgNNP (100 μL, 5 μg mL -1 ) The wound was treated once (only once), and the wound surface was photographed on days 0, 2, 4, 6, 8, and 10, and the mice were monitored. Fig. 9 (a). It can be seen that the quantitative results prove that the wounds of all groups have scabbed on the 4th day, and all wounds have shrunk to varying degrees. Data analysis was used to analyze the remaining wound size using the image analysis program image.J. The wound sizes of the different treatment groups began to differ after 6 days of treatment. The results showed that the remaining wound size of the CEP-POP-AgNNP group was only 13.34±0.57% (control group = 41.34±0.57%, CEP-POP = 19.1%, AgNNPs = 16%). At the same time, compared with the other groups, the CEP-POP-AgNNP group had significant wound closure on the 8th day, with a healing rate of 89.34±0.57%, much higher than the other groups. After 10 days of treatment, the wounds in the CEP-POP-AgNNP group were basically healed, with a tissue reconstruction rate of 98.34±0.57%, while 14.3±0.57%, 7.33±1.15%, and 5.1% of the wounds in the PBS group, AgNNPs group, and CEP-POP group remained open, respectively. These results further indicate that compared with the other groups, CEP-POP-AgNNP can not only significantly reduce bacterial invasion of wounds, but also significantly accelerate wound healing. At the same time, the body weight of each mouse was recorded from day 0 to day 10 to investigate the health status of the mice. Fig. 9 As shown in (c), the body weight of mice decreased slightly only on the first day of anesthesia. At other times, there was no significant difference in the body weight of mice in different groups.

[0084] To further evaluate the wound healing process after bacterial eradication, skin samples collected around the infected wounds were stained with hematoxylin and Masson's trichrome to provide more reliable details ( Fig.10 ). For the control group (PBS solution), CEP-POP and AgNNPs, obvious scabs and incomplete skin tissues were observed in the mice, which was consistent with the digital photos of the wounds, indicating that the wounds were not completely closed. In addition, severe subcutaneous bleeding (red frame) may also occur in the control group. While the degree of healing of mice in the CEP-POP, AgNNPs and CEP-POP-AgNNP groups was different, they showed similar tissue regeneration, structural arrangement and abundant capillary distribution to provide nutrition (blue frame), indicating that skin tissue regeneration is rapid. The CEP-POP-AgNNP group also showed thicker epithelialized areas of skin tissue and a large number of hair follicles (yellow frame). Masson's trichrome staining showed a large amount of regeneration and distribution of collagen fibers in different treatment groups.

[0085] In a BALB / C mouse model of Staphylococcus aureus infection on the back wound, the CEP-POP-AgNNP group was found to lead to complete wound healing on day 10. Toxicological studies confirmed that CEP-POP, AgNNPs, and CEP-POP-AgNNPs had no obvious inflammatory damage or morphological damage to major organs (heart, liver, spleen, kidney, and lung) ( Fig.11 ), in order to verify this speculation, routine blood tests were performed on mice. Fig.12 As shown, the RBC, HGB, WBC, MCV, MCHC, HCT, MCH, PLT, and blood indexes of healthy mice after anesthesia (the blank group was healthy mice without established animal model) were all within the normal fluctuation range, indicating that the synergistic strategy is safe for mice and has no obvious toxicity.

[0086] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing a silver-based porous organic framework material, characterized in that: The following steps are involved: (1) Preparation of 4,4′-diformyldibenzo-18-crown-6: dibenzo-18-crown-6, trifluoroacetic acid and hexamethylenetetramine were mixed, stirred and heated for reaction under a protective atmosphere, and after the reaction was completed, the mixture was cooled to obtain a yellow-brown precipitate, which was washed, filtered and dried to obtain 4,4′-diformyldibenzo-18-crown-6; (2) Preparation of CEP-POP: Add redistilled pyrrole, 4,4′-diformyldibenzo-18-crown-6 obtained in step (1) and FeCl2·4H2O to propionic acid, reflux under a protective atmosphere, wash and dry the obtained black powder to obtain CEP-POP; (3) Preparation of CEP-POP-AgNNP: CEP-POP and SNP obtained in step (2) are added to water to obtain a mixed solution, which is stirred at room temperature overnight. After centrifugation, the obtained precipitate is washed and freeze-dried to obtain SNP@CEP-POP. Silver nitrate solution is added to the obtained SNP@CEP-POP, and the mixture is stirred at room temperature for reaction. The solid obtained by the stirring reaction is washed and dried to obtain a porous organic framework material, namely CEP-POP-AgNNP.

2. The preparation method according to claim 1, characterized in that: In step (1), the ratio of the added amounts of dibenzo-18-crown-6-ether, hexamethylenetetramine and trifluoroacetic acid is 10 mmol:10 mmol:7 mL.

3. The preparation method according to claim 1, characterized in that: In step (1), the protective atmosphere is argon; the temperature of the heating reaction is 90° C., and the time of the heating reaction is 12 to 15 hours.

4. The preparation method according to claim 1, characterized in that: In step (2), the ratio of the added amounts of redistilled pyrrole, 4,4′-diformyldibenzo-18-crown-6, FeCl2·4H2O and propionic acid is 4mmol:4mmol:1.2mmol:120mL.

5. The preparation method according to claim 1, characterized in that: In step (2), the protective atmosphere is argon; the temperature of the reflux reaction is 140° C., and the time of the reflux reaction is 48 h.

6. The preparation method according to claim 1, characterized in that: In step (3), the mass ratio of CEP-POP to SNP is 1:12; and the concentration of CEP-POP in the mixed solution is 2.5 mg / mL.

7. The preparation method according to claim 1, characterized in that: In step (3), the mass ratio of SNP@CEP-POP to silver nitrate solution is 65:68; the concentration of silver nitrate solution is 1×10 -3 M; the stirring reaction time is 3h.

8. The silver-based porous organic framework material obtained by the preparation method according to any one of claims 1 to 7.

9. Use of the silver-based porous organic framework material according to claim 8 in the preparation of drugs against Gram-negative bacteria and Gram-positive bacteria.

10. The use according to claim 9, characterized in that: The Gram-negative bacteria is Escherichia coli; the Gram-positive bacteria is Staphylococcus aureus.

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