Covalent organic framework polymer capable of selectively inactivating staphylococcus aureus and application of covalent organic framework polymer

Abstract

The invention discloses a covalent organic framework polymer capable of selectively inactivating staphylococcus aureus and application of the covalent organic framework polymer. The covalent organic framework polymer is obtained by polymerization of a ruthenium complex and heteropoly acid through aminal reaction, and the ruthenium complex is bis (hexafluorophosphate) tris (5, 5 '-bis (diethoxymethyl)-2, 2'-dipyridyl) ruthenium; the preparation method comprises the following steps: adding bis (hexafluorophosphate) tris (5, 5 '-bis (diethoxymethyl)-2, 2'-dipyridyl) ruthenium and Tris-V6O19 into an organic solvent, and carrying out solvothermal reaction, so as to obtain the covalent organic framework polymer. The m-RuV-COF prepared by the invention can selectively inactivate staphylococcus aureus at an extremely low concentration, and has a very small inactivation effect on escherichia coli. And a new effective strategy is provided for specifically solving the problem of staphylococcus aureus infection.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to covalent organic framework polymers that selectively inactivate Staphylococcus aureus and their applications. Background Technology

[0002] Bacterial infections pose a serious threat to human health in fields such as medicine, food, and the environment. Staphylococcus aureus, as a representative of Gram-positive bacteria, is a major pathogen causing various diseases. Traditional broad-spectrum antibacterial therapy rapidly controls infection symptoms by indiscriminately inhibiting or killing multiple bacterial pathogens; however, this treatment process has significant drawbacks. Due to its lack of specificity, broad-spectrum antibacterial therapy not only eliminates pathogenic bacteria but also disrupts the beneficial symbiotic flora colonizing the human gut, skin, and other areas. More seriously, the overuse of broad-spectrum antibacterial drugs can induce adaptive gene mutations in bacteria, causing previously sensitive strains to gradually evolve into multidrug-resistant bacteria, leading to a situation where no effective drugs are available for clinical treatment. Therefore, the development of novel materials that combine high selectivity and efficient bactericidal capabilities is urgently needed.

[0003] Currently, novel antibacterial modalities mainly include photothermal therapy (PTT) and photodynamic therapy (PDT). PTT utilizes specific wavelengths of light (such as blue light, ultraviolet light, or near-infrared light) combined with photosensitizing substances or direct irradiation to destroy the structure of microorganisms such as bacteria, fungi, or viruses through photochemical reactions. PDT involves irradiating the body with specific wavelengths of light after a photosensitizer enters the body. When the photosensitizer absorbs the light, a photochemical reaction occurs, generating reactive oxygen species that inhibit bacteria, thus achieving a therapeutic effect. While PDT avoids the problem of chemical drug residues, the penetration efficiency of the photosensitizers it relies on against Gram-positive bacteria is generally less than 30%. This is mainly due to the 20-80 nm thick peptidoglycan layer structure unique to Gram-positive bacteria such as Staphylococcus aureus, which forms a physical barrier that significantly hinders the effective penetration of reactive oxygen free radicals. Experimental data shows that conventional phototherapy can achieve a 99% kill rate against Escherichia coli (Gram-negative), but only achieves less than 50% clearance against Staphylococcus aureus (Gram-positive).

[0004] In the fields of modern medicine and microbial control, achieving selective inactivation of Staphylococcus aureus, a typical Gram-positive bacterium, and even a wider range of Gram-positive bacteria, faces severe and complex challenges. Currently, antibacterial materials with photothermal or photodynamic effects are broad-spectrum materials, exhibiting high kill rates against both Gram-negative bacteria like Escherichia coli and Gram-positive bacteria like Staphylococcus aureus. Broad-spectrum sterilization disrupts the normal flora environment; while killing the target bacterium, other bacteria are also killed. Selective sterilization, on the other hand, selectively kills pathogens while avoiding disruption of the normal human flora, reducing the risk of secondary infections, and avoiding the "indiscriminate attack" of broad-spectrum antibiotics. Designing sterilization mechanisms targeting specific pathogens (such as drug-resistant Staphylococcus aureus) reduces selective pressure on unrelated bacteria and slows the spread of resistance. Therefore, a material with selective inactivation capabilities for Staphylococcus aureus is needed to selectively and effectively kill the bacterium at low concentrations, promoting the development of precision sterilization. Summary of the Invention

[0005] In view of the above-mentioned prior art, the purpose of this invention is to provide a covalent organic framework polymer with selective inactivation of Staphylococcus aureus and its applications. This invention first prepares the ruthenium complex bis(hexafluorophosphate)tris(5,5'-bis(diethoxymethyl)-2,2'-bipyridine)rutheniumRu(bpy-oEt)3(PF6)2, and then reacts it with the heteropolyacid tris-V6O 19 Polymerization yields a covalent organic framework polymer m-RuV-COF that selectively inactivates Staphylococcus aureus. It can be used for combined antibacterial activity through various methods, exhibits excellent biocompatibility, and has no toxic side effects.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A first aspect of the present invention provides a covalent organic framework polymer having the selective inactivation effect on Staphylococcus aureus, said covalent organic framework polymer being obtained by polymerization of a ruthenium complex and a heteropolyacid via an acetal-amine reaction, wherein the ruthenium complex is bis(hexafluorophosphate)tris(5,5'-bis(diethoxymethyl)-2,2'-bipyridine)ruthenium; and the heteropolyacid is Tris-V6O. 19 .

[0007] Preferably, the covalent organic framework polymer is prepared by the following method: bis(hexafluorophosphate)tris(5,5'-bis(diethoxymethyl)-2,2'-bipyridine)ruthenium and Tris-V6O 19 A solvothermal reaction was carried out by adding an organic solvent to obtain a covalent organic framework polymer that selectively inactivates Staphylococcus aureus.

[0008] Preferably, the bis(hexafluorophosphate)tris(5,5'-bis(diethoxymethyl)-2,2'-bipyridine)ruthenium and Tris-V6O 19 The molar ratio is 1:2.

[0009] Preferably, the organic solvent is obtained by mixing trimethylbenzene, 1,4-dioxane and glacial acetic acid in a volume ratio of 2:2:0.8.

[0010] Preferably, the temperature of the solvothermal reaction is 180°C and the time is 72 hours.

[0011] Preferably, the bis(hexafluorophosphate)tris(5,5'-bis(diethoxymethyl)-2,2'-bipyridine)ruthenium is prepared by the following method: 2,2'-bipyridine-5,5'-dicarboxaldehyde and ruthenium trichloride trihydrate were added to ethanol under a protective atmosphere and refluxed. A saturated ammonium hexafluorophosphate solution was added, and the mixture was filtered and washed to obtain an orange-red solid, namely bis(hexafluorophosphate)tris(5,5'-bis(diethoxymethyl)-2,2'-bipyridine)ruthenium.

[0012] Preferably, the molar ratio of 2,2'-bipyridine-5,5'-dicarboxaldehyde to ruthenium trichloride trihydrate is 3.1:1.

[0013] Preferably, the reflux reaction is carried out at a temperature of 80°C for 72 hours.

[0014] A second aspect of the invention provides the use of covalent organic framework polymers in the preparation of medicaments having selective inactivation effects on Gram-positive bacteria, wherein the Gram-positive bacteria are Staphylococcus aureus.

[0015] Preferably, when the drug concentration is 125 μg / mL, the survival rate of Staphylococcus aureus is 3.25% and the survival rate of Escherichia coli is 70.08%.

[0016] The beneficial effects of this invention are: (1) Preparation of ruthenium complex Ru(bpy-oEt)3(PF6)2 and heteropolyacid Tris-V6O 19 The resulting m-RuV-COF exhibits excellent photothermal conversion under 638nm laser irradiation and possesses POD-like enzyme activity, capable of converting endogenous H2O2 in the infection microenvironment into toxic ·OH. At extremely low concentrations, it can selectively inactivate Staphylococcus aureus, but its inactivation effect on Escherichia coli is minimal. This provides a new and effective strategy for specifically addressing Staphylococcus aureus infections.

[0017] (2) The preparation method of the present invention is simple, the prepared m-RuV-COF has good biocompatibility, a very low hemolysis rate of red blood cells, minimal impact on the viability of 3T3 cells, and no toxic side effects on the human body. It provides a new strategy for developing new materials with both high selectivity and high efficiency bactericidal ability and for the precise treatment of Gram-positive bacterial infections. Attached Figure Description

[0018] Figure 1 : (a) Ru(bpy-oEt)3(PF6)2、Tris-V6O 19 (a) Fourier transform infrared spectra of m-RuV-COF; (b) Fourier transform infrared spectra of m-RuV-COF and p-RuV-COF; (c) Fourier transform infrared spectra of m-RuV-COF. 13 (d) Weight variation of m-RuV-COF under nitrogen atmosphere with increasing temperature; (e) Low-temperature N2 absorption isotherm of m-RuV-COF; (f) Pore size distribution curve of m-RuV-COF; (g) Low-temperature N2 absorption isotherm of p-RuV-COF; (h) Pore size distribution curve of p-RuV-COF; (i) XPS spectrum of m-RuV-COF; (j) XPS spectrum of V 2p; (k) XPS spectrum of C 1s; (l) High-resolution XPS spectrum of Ru 3p; (m) XPS spectrum of N 1s; (n) XPS spectrum of P 2p; (o) XPS spectrum of F 1s; (p) High-resolution XPS spectrum of O 1s; (q) XRD pattern of m-RuV-COF; Figure 2 (a) SEM of m-RuV-COF at a scale of 500 nm; (b) SEM of m-RuV-COF at a scale of 500 nm; (c) TEM of m-RuV-COF at a scale of 100 nm; (d) TEM of m-RuV-COF at a scale of 20 nm; (e) TEM of p-RuV-COF at a scale of 100 nm; (f) TEM of p-RuV-COF at a scale of 20 nm; (g) HR-TEM of m-RuV-COF at a scale of 1 nm; (h) HR-TEM of p-RuV-COF at a scale of 1 nm; (i) HADDF-STEM image of m-RuV-COF at a scale of 200 nm; and elemental distribution maps of C, N, O, F, P, Ru, and V in m-RuV-COF at a scale of 500 nm. Figure 3(a) Temperature curves of m-RuV-COF at different power densities; (b) Temperature curves of m-RuV-COF at different concentrations; (c) Corresponding infrared thermal images of m-RuV-COF at different concentrations; (d) m-RuV-COF aqueous solution (125 μg / mL) under four laser beams (λ = 638 nm, 1.5 W / cm²). -2 Temperature change curves during irradiation on / off cycles; (e) m-RuV-COF (125 μg / mL) at 638 nm, 1.5 W / cm 2 (f) Temperature change curve after laser irradiation; (g) Photothermal conversion efficiency of m-RuV-COF; (g) Comparison of temperature of m-RuV-COF under 638nm laser (1.5 W / cm2) irradiation with the temperature of the freshly prepared aqueous solution after 30 days of incubation in water.

[0019] Figure 4 (a) UV absorption of each group of m-RuV-COF near 650 nm; (b) UV absorption of each group of p-RuV-COF near 650 nm; (c) UV absorption of m-RuV-COF at 650 nm under illumination or without illumination; (d) UV absorption of p-RuV-COF at 650 nm under illumination or without illumination; (e) The production of 200 μg / mL m-RuV-COF solution in PBS at different pH values. . UV absorption of OH; (f) Generation of p-RuV-COF solution at a concentration of 200 μg / mL in PBS at different pH values. . UV absorption of OH; (g) UV absorption peaks at 650 nm (pH 5.5) of m-RuV-COF solutions of different concentrations; (h) UV absorption peaks at 650 nm (pH 5.5) of p-RuV-COF solutions of different concentrations. Figure 5 (a) Photographs of bacterial colonies formed by Staphylococcus aureus after different treatments; (b) Survival rate of Staphylococcus aureus after different treatments; (c) Biofilm removal rate of Staphylococcus aureus after different treatments. Figure 6 (a) Transmission electron micrographs of Staphylococcus aureus and Escherichia coli under different treatments, scale bar 2.0 μm; (b) Fluorescence images of Staphylococcus aureus and Escherichia coli after co-staining with various treatments, scale bar 100 μm; Figure 7(a) Hemolysis of different concentrations of m-RuV-COF; (b) MTT assay of 3T3 cells after 24 h of treatment with different concentrations of m-RuV-COF; (c) Cell scratch healing rate; (d) Cell migration assay by wound healing. Figure 8 (a) Photographs of the back wounds of mice infected with Staphylococcus aureus after treatment in each group; (b) Changes in mouse body weight during treatment; (c) Curves showing changes in wound area over time; Figure 9 H&E and Masson trichrome staining images of wound tissues in each group on day 8 of wound healing; Figure 10 H&E staining of the major organs of mice: heart, liver, spleen, lungs and kidneys. Detailed Implementation

[0020] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0021] As introduced in the background section, traditional broad-spectrum antibacterial therapy indiscriminately inhibits or kills a variety of bacterial pathogens, lacking specificity in its action. Overuse of broad-spectrum antibiotics can induce adaptive gene mutations in bacteria, causing previously sensitive strains to gradually evolve into multidrug-resistant bacteria, leading to a situation where no effective drugs are available in clinical treatment. Therefore, the development of novel materials that combine high selectivity and efficient bactericidal capabilities is urgently needed. Currently, although ruthenium complexes or Tris-V6O... 19 There are reports of its use in the preparation of antibacterial drugs, but the drugs prepared are all broad-spectrum antibacterial drugs and cannot achieve selective bactericidal effects.

[0022] Based on this, the object of the present invention is to provide a covalent organic framework polymer with selective inactivation of Staphylococcus aureus and its applications. The present invention uses the ruthenium complex bis(hexafluorophosphate)tris(5,5'-bis(diethoxymethyl)-2,2'-bipyridine)rutheniumRu(bpy-oEt)3(PF6)2 and the heteropolyacid Tris-V6O 19 The covalent organic framework polymer m-RuV-COF was prepared from a monomer via an acetal-amine reaction. Studies revealed that only the ruthenium complex bis(hexafluorophosphate)tris(5,5'-bis(diethoxymethyl)-2,2'-bipyridine)ruthenium reacts with Tris-V6O. 19Only polymers obtained through polymerization possess the ability to selectively inactivate Staphylococcus aureus. The ruthenium complex bis(hexafluorophosphate)tris(5,5'-bis(diethoxymethyl)-2,2'-bipyridine)ruthenium Ru(bpy-oEt)3(PF6)2 is obtained by reacting 2,2'-bipyridine-5,5'-dicarboxaldehyde, ruthenium trichloride, and anhydrous ethanol; while bis(hexafluorophosphate)hexa(2-formylpyridine)ruthenium Ru(bpy-CHO)3(PF6)2 is prepared from 2,2'-bipyridine-4,4'-dicarboxaldehyde and ruthenium trichloride. The 2,2'-bipyridine-5,5'-dicarboxaldehyde used to prepare bis(hexafluorophosphate)tris(5,5'-bis(diethoxymethyl)-2,2'-bipyridine)ruthenium is an isomer of the 2,2'-bipyridine-4,4'-dicarboxaldehyde used to prepare bis(hexafluorophosphate)hexa(2-formylpyridine)ruthenium. Although Ru(bpy-oEt)3(PF6)2 and Ru(bpy-CHO)3(PF6)2 have different structures and are not isomers, they are related by the fact that bis(hexafluorophosphate)tris(5,5'-bis(diethoxymethyl)-2,2'-bipyridine)ruthenium reacts with Tris-V6O 19 During polymerization, an acidic environment is present, causing the acetal structure in the molecule to hydrolyze and transform into an aldehyde group. Therefore, the resulting m-RuV-COF and p-RuV-COF are structural isomers. However, bis(hexafluorophosphate)hexa(2-formylpyridine)ruthenium ...2-formylpyridine)hexa(formylpyridine)ruthenium bis(2-formylpyridine)hexa(formylpyridine)hexa(formylpyridine)hexa(formylpyridine)hexa(formylpyridine)hexa(formylpyridine)hexa(formylpyridine)hexa(formylpyridine)hexa(formylpyridine)hexa(formylpyridine)hexa(formylpyridine)hexa(formylpyridine)hexa(formylpyridine)hexa(formylpyridine)hexa(formylpyridine)hexa(formylpyridine)hexa(formylpyridine)hexa(formylpyridine)hexa(formylpyridine)hexa(formylpyridine)hexa(formylpyridine)hexa(formylpy 19 The polymer p-RuV-COF obtained by polymerization does not have the ability to selectively inactivate Staphylococcus aureus.

[0023] The aldehyde group formed after hydrolysis of Ru(bpy-oEt)3(PF6)2 is located ortho-to the pyridine N group, while the aldehyde group of Ru(bpy-CHO)3(PF6)2 is located para-to the pyridine N group. The ortho-to position of the aldehyde group compared to the para-position results in greater steric hindrance and a more crowded steric environment in the ruthenium complex, thus making the spatial structure of Ru(bpy-oEt)3(PF6)2 more distorted than that of Ru(bpy-CHO)3(PF6)2. This leads to a more complex steric structure in Ru(bpy-oEt)3(PF6)2 with the heteropolyacid Tris-V6O. 19 The prepared m-RuV-COF is more effective than Ru(bpy-CHO)3(PF6)2 with the heteropolyacid Tris-V6O 19 The prepared p-RuV-COF has a more developed pore structure, larger pores, and better mass transfer, making it easier to disrupt the cell membrane of Staphylococcus aureus and thus selectively inhibit Staphylococcus aureus.

[0024] The m-RuV-COF prepared in this invention can convert H2O2 into ·OH with peroxidase-like (POD) activity under acidic conditions. It can also adhere to the bacterial surface via electrostatic interactions, and then achieve localized heating through photothermal conversion, thereby inactivating enzymes and disrupting the physiological structure of the bacteria. Simultaneously, it exhibits negligible hemolytic activity and minimal damage to normal cells at extremely low concentrations. Furthermore, while achieving a bactericidal rate of over 95% against Staphylococcus aureus, its bactericidal rate against Escherichia coli is less than 30%, indicating its great potential for highly selective and efficient bactericidal action.

[0025] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0026] The test materials used in the embodiments of this invention are all conventional test materials in the art and can be purchased through commercial channels.

[0027] Example 1: Preparation of m-RuV-COF (1) Preparation of Ru(bpy-oEt)3(PF6)2: In a 100 mL round-bottom flask, 2,2'-bipyridine-5,5'-dicarboxaldehyde (658 mg, 3.1 mmol) was added to ruthenium trichloride trihydrate (261.4 mg, 1.0 mmol) dissolved in 50 mL of anhydrous ethanol. The mixture was stirred and heated to 80 °C under nitrogen atmosphere and refluxed for 72 h, then cooled to room temperature. Unreacted solids were removed by filtration. The solution was concentrated to approximately 5 mL under reduced pressure, and then 5 mL of saturated ammonium hexafluorophosphate solution (saturated NH4PF6 solution) was added dropwise with stirring (5 mL / min). A rapid orange-red precipitate formed. After filtration, the precipitate was washed with cold water and diethyl ether, and then dried under vacuum to obtain an orange-red solid, Ru(bpy-oEt)3(PF6)2, with a yield of 75%. The synthetic route is as follows: .

[0028] (2) Tris-V6O 19 Preparation: Vanadylamine (1.63 g, 13.9 mmol), 2-amino-2-(hydroxymethyl)-1,3-propanediol (0.83 g, 6.9 mmol), hydrazine hydrate (80%, 180 μL), and 20 mL of deionized water were placed in a 50 mL reactor and heated at 210 °C for 24 h, then cooled to room temperature. The resulting solution was filtered and left at room temperature for 7 days. The product deposited from the filtrate was black cubic crystals, which, upon filtration, yielded black crystals, namely Tris-V6O. 19The molecular formula is (NH4)2[V IV 3V V 3O 10 [{NH2C(CH2O)3}3], the structural formula is: .

[0029] (3) Preparation of m-RuV-COF 177.15 mg Ru(bpy-oEt)3(PF6)2 (0.15 mmol) and 224.42 mg TrisV6O9 (0.3 mmol) were placed in a 25 mL reaction vessel, followed by the addition of 2.0 mL mesitylene, 2.0 mL 1,4-dioxane, and 0.8 mL glacial acetic acid. The mixture was purged with N2 for approximately 5 min. The reaction vessel was then placed in an oven and heated to 180 °C for 72 h. After cooling, the mixture was filtered and washed successively with methanol, dichloromethane, and DMF, and then dried under vacuum at 100 °C for 12 h to obtain a black powder, m-RuV-COF, with a yield of 54%. The structural formula of m-RuV-COF is: .

[0030] Comparative Example: Preparation of p-RuV-COF The difference from Example 1 lies in step (3), where Ru(bpy-oEt)3(PF6)2 is replaced with an equimolar amount of Ru(bpy-CHO)3(PF6)2, ultimately yielding black powder p-RuV-COF with a yield of 58%. The structural formula of p-RuV-COF is: .

[0031] The preparation method of Ru(bpy-CHO)3(PF6)2 is as follows: In a 100 mL round-bottom flask, 2,2'-bipyridine-4,4'-dicarboxaldehyde (700.2 mg, 3.1 mmol) was added to ruthenium trichloride trihydrate (261.4 mg, 1 mmol) dissolved in 10 mL ethanol and 5 mL H₂O. The mixture was stirred and heated to 80 °C under nitrogen atmosphere and refluxed for 20 h, then cooled to room temperature. Unreacted solids in the reaction solution were removed by filtration. Ethanol was removed by rotary evaporation to obtain the crude product. The mixture was collected and redispersed in 25 mL H₂O, and undissolved impurities were removed by filtration. A saturated NH₄PF₆ solution was added dropwise with stirring (5 mL / min), and an orange precipitate appeared rapidly. After filtration, the precipitate was washed with H₂O and then dried under vacuum to obtain an orange solid, Ru(bpy-CHO)₃(PF₆)₂, with a yield of 52%. The synthetic route is as follows: .

[0032] Example 2: Characterization (1) The structure of m-RuV-COF was analyzed using infrared spectroscopy. For example... Figure 1 As shown in (a), the CO stretching vibration and Tris-V6O in the acetal structure of Ru(bpy-oEt)3(PF6)2 19 The stretching vibration of NH in the amino group is significantly weakened, at 1644 cm⁻¹. -1 A clear vibrational peak can be observed at the point, corresponding to the stretching vibration of the newly formed CN group. These results indicate that the acetal group (-O-CH(O)-) and the amino group (-NH2) have successfully undergone a polymerization reaction. Figure 1 (b) also shows that p-RuV-COF has a -C=N peak, which proves that the material has been successfully polymerized.

[0033] Through solid 13 C-NMR further confirmed the carbon skeleton structure of the m-RuV-COF complex. Figure 1 As shown in (c), the chemical shift at 66 ppm belongs to carbon in CO, the carbon signal at 165 ppm belongs to carbon in C=N, and the peak at 149 ppm is attributed to aromatic carbon in the m-RuV-COF structure.

[0034] Figure 1 (d) shows the thermogravimetric curve of m-RuV-COF, and the thermogravimetric analysis demonstrates the thermal stability of m-RuV-COF. The weight decrease below 100℃ is due to the volatilization of adsorbed water or residual organic solvents in the material. Up to 500℃, the weight of m-RuV-COF remains above 50%.

[0035] The porosity of m-RuV-COF and p-RuV-COF was estimated using low-temperature N2 adsorption-desorption experiments. Figure 1 (e) and Figure 1As shown in (f), both m-RuV-COF and p-RuV-COF exhibit significant hysteresis loops. Adsorption capacity increases slowly at low P / P0 and rapidly in the medium-to-high P / P0 range, consistent with the adsorption behavior of mesoporous materials. They exhibit typical type IV isotherm characteristics, indicating that both materials are mesoporous, with mesopores playing a crucial adsorption role. The comparison shows that m-RuV-COF generally has a higher adsorption capacity than p-RuV-COF at the same P / P0, suggesting that m-RuV-COF has a larger specific surface area or pore volume, resulting in a stronger adsorption and storage capacity for N2. Furthermore, the hysteresis loop morphology differs between the two materials: m-RuV-COF has a wider hysteresis loop (larger adsorption and desorption branching), while p-RuV-COF has a relatively narrower hysteresis loop. This suggests that m-RuV-COF has a more complex pore structure, potentially with a wider mesopore size distribution or larger pore shape, while p-RuV-COF has relatively regular channels and a more concentrated pore size distribution. Calculations show that the BET surface area of ​​m-RuV-COF is 37.2860 m² / g, and its porosity is 0.092500 cm³ / g. The BET surface area of ​​p-RuV-COF is 10.2561 m² / g, and its porosity is 0.064483 cm³ / g. These results are consistent with the characteristics identified by the adsorption isotherms. Figure 1 (g) and Figure 1 The BJH models of m-RuV-COF and p-RuV-COF in (h) also show that both materials are predominantly mesoporous.

[0036] The surface elemental composition of m-RuV-COF was investigated using X-ray photoelectron spectroscopy (XPS). Figure 1 (i) shows the measured spectrum of m-RuV-COF, with clear peaks for C, N, O, F, P, Ru and V, with atomic proportions of 5.43%, 3.46%, 7.15%, 2.15%, 1.38%, 0.57% and 79.85%, respectively. Figure 1 (j) shows the V 2p spectrum of m-RuV-COF. Four peaks were fitted at 515.4, 516.6, 523, and 524.1 eV, corresponding to V2, respectively. Ⅴ 2p 1 / 2 V Ⅳ 2p 1 / 2 V Ⅴ 2p 2 / 3 V Ⅳ 2p 2 / 3 ; Figure 1 (k) shows the C 1s spectrum of m-RuV-COF, with four peaks at 284.2, 284.8, 285.7, and 288, corresponding to C(sp... 2 ), C (sp 3 ), CO and C=N; Figure 1 (l) shows the Ru 3p spectrum of m-RuV-COF, with two peaks at 462.2 and 484.4, which are attributed to Ru 3p. 2 / 3 、Ru 3p 1 / 2 ; Figure 1 (m) is the N 1s spectrum of m-RuV-COF, which is divided into two peaks at 399.9 and 401.4 eV, assigned to C=N and NH4, respectively. + ; Figure 1 (n) is the p 2p spectrum of m-RuV-COF. Figure 1 (o) represents the F 1s spectrum of m-RuV-COF, all attributed to PF6. - ; Figure 1 (p) is the O 1s spectrum of m-RuV-COF, with peaks at 529.7 and 531.3 eV attributed to absorbed -OH and lattice oxygen in heteropolyacids, respectively.

[0037] Powder X-ray diffraction (XRD) was performed to study the crystallinity of m-RuV-COF, such as... Figure 1 As shown in (q), the XRD results show two distinct peaks at 8.8° and 17.8°. The main peak is relatively sharp, indicating crystallinity. However, the background noise level is high, suggesting that the material may not be highly crystallizable.

[0038] (2) The surface morphology of the synthesized m-RuV-COF was studied using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). For example... Figure 2 (a) ~ Figure 2 As shown in (b), the synthesized m-RuV-COF consists of a tightly stacked layered structure. Figure 2 (c)~ Figure 2 (d) TEM shows that the layered structure of m-RuV-COF becomes clearer with increasing scale. Figure 2 (e)~ Figure 2 (f) TEM also shows the layered structure of p-RuV-COF. Figure 2 (g) and Figure 2 (h) is a high-resolution transmission electron microscope (HR-TEM), which reveals lattice fringes with spacings of 0.288 and 0.210 nm in m-RuV-COF and 0.152 and 0.106 nm in p-RuV-COF, indicating that both materials formed crystalline structures during polymerization, but the crystalline structures differ. Figure 2In (i), the high-angle annular dark-field scanning TEM (HAADF-STEM) also allows for direct observation of the layered structure of interconnected macropores in m-RuV-COF. Meanwhile, elemental mapping shows that O, F, P, Ru, C, and V are uniformly distributed on m-RuV-COF.

[0039] (3) The photothermal effect of the material was measured by irradiating m-RuV-COF with a 638nm laser for 10min. Figure 3 (a) shows the results at 0, 0.5, 1.0, 1.2, and 1.5 W / cm², respectively. 2 Laser power-dependent photothermal effect of m-RuV-COF under 638nm laser irradiation. Figure 3 (e) are thermal images of the heating process of aqueous solutions of m-RuV-COF with different concentrations (0-150 μg / mL) over 10 min; Figure 3 (b) is 1.5 W / cm 2 The concentration-dependent photothermal effect of m-RuV-COF under 638 nm laser irradiation showed that as the concentration of m-RuV-COF increased, the temperature change caused by the heating of its suspension became increasingly larger. Figure 3 (d) is the 638nm laser at 1.5 W / cm². 2 The temperature change curves of m-RuV-COF (125 μg / mL) after four light-cooling cycles are shown below. The temperature change of m-RuV-COF was recorded using an ON / OFF cyclic irradiation experiment. The results show that after four cycles (638 nm, 1.5 W / cm²), m-RuV-COF exhibits optimal temperature performance. 2 The sample exhibited good photothermal stability, with little change in the temperature rise and cooldown trends. These results indicate that m-RuV-COF possesses good photothermal effects and stability, making it a potential photothermal antibacterial agent. Maintaining a fixed m-RuV-COF concentration of 125 μg / mL while varying the laser power revealed that the temperature rise continued to increase with increasing laser power. These results demonstrate that m-RuV-COF exhibits high photothermal conversion efficiency and concentration-dependent photothermal performance. Figure 3 (g) is an aqueous dispersion of m-RuV-COF (125 μg / mL) irradiated with a 638 nm laser (1.5 W / cm²). 2 Photothermal effect. Figure 3 (f) shows τ in the formula for calculating photothermal conversion efficiency. S Based on the above values, m-RuV-COF (125 µg / mL) at 638 nm (1.5 W / cm²) corresponds to the θ value. 2 The calculated photothermal conversion efficiency is 54.8%.

[0040] (4) Hydroxyl radical generation capacity test Three solutions were prepared: TMB, H2O2 + TMB, and TMB + H2O2 + m-RuV-COF. The concentrations of TMB and H2O2 in each solution were 75 μL / mL, and the concentration of m-RuV-COF was 200 μg / mL. The total volume of each solution was 1 mL, and the volume was brought up to 1 mL with PBS at pH 5.5. The UV absorbance of each solution was measured. To verify whether light affects the enzyme activity of m-RuV-COF, a 200 μg / mL aqueous solution of m-RuV-COF was prepared, and its UV absorbance was measured before and after light exposure and plotted. 200 μg / mL solutions of m-RuV-COF were prepared, and the volume was brought up to 1 mL with PBS at pH values ​​of 1.5, 2.5, 3.5, 4.5, 5.5, and 6.5, respectively, and their UV absorbance was measured. Prepare 1 mL solutions of m-RuV-COF with concentrations of 50, 100, 150, 200, 250, and 300 μg / mL using PBS at pH 5.5, and measure their UV absorption at different concentrations under the same pH conditions. The detection method for p-RuV-COF is the same as described above.

[0041] like Figure 4 As shown in (a), m-RuV-COF only induces TMB to turn the solution blue and exhibits a prominent UV absorption peak at 650 nm in the presence of H2O2. However, m-RuV-COF alone or H2O2 alone does not induce oxidation. This result indicates that m-RuV-COF has excellent POD-like activity. Figure 4 The results shown in (b) are similar to those of m-RuV-COF, indicating that p-RuV-COF also has excellent POD-like activity. However, the UV absorption peak is much lower than that of m-RuV-COF, indicating that m-RuV-COF has stronger POD-like activity. Figure 4 (c) and Figure 4 (d) shows that the signal intensity is further enhanced after laser irradiation, indicating that photothermal therapy can work synergistically with enzyme therapy to cause severe oxidative damage to various bacteria. Figure 4 (e) shows the changes in enzyme activity of m-RuV-COF at different pH values. It can be seen that the enzyme activity of m-RuV-COF first increases and then decreases with the change of pH value, reaching the optimal value at pH 3.5. In the infection environment (pH 5.5), m-RuV-COF also has significant catalytic activity, which can convert low concentrations of H2O2 into toxic ·OH, thereby effectively killing bacteria. Figure 4(f) also shows that the enzyme activity of p-RuV-COF is optimal at pH 5.5, but is still lower than that of m-RuV-COF. Figure 4 (g) and (h) show that the enzyme activities of both m-RuV-COF and p-RuV-COF are concentration-dependent. With increasing concentration (pH 5.5), the enzyme activity significantly increases, and the enzyme activity of m-RuV-COF at all concentrations is higher than that of p-RuV-COF at the same concentration. These results indicate that both m-RuV-COF and p-RuV-COF possess POD-like activity, but m-RuV-COF exhibits stronger enzyme activity.

[0042] Test Example 1: In vitro antibacterial performance test Staphylococcus aureus and Escherichia coli were used as representatives of Gram-positive and Gram-negative bacteria, respectively, to test the antibacterial properties of m-RuV-COF and p-RuV-COF.

[0043] The experiment was divided into 6 groups: (I) PBS, (II) m-RuV-COF, (III) m-RuV-COF + laser, (IV) m-RuV-COF + H2O2, (V) m-RuV-COF + H2O2 + laser, and (VI) p-RuV-COF + H2O2 + laser. The total volume of the mixed solution in each group was 1 mL, and the bacterial concentration was 100 μL / mL (colony count was 10^6). 8 CFU mL -1 The concentrations of m-RuV-COF and p-RuV-COF were 125 μg / mL, and the laser power was 1.5 W / cm². 2 (638 nm, 10 min), H2O2 concentration 10 mmol / mL. The mixtures from each group were incubated in a shaker at 37°C for 24 h. 80 μL of each mixture was then transferred to solid culture medium (10 g LB broth + 6 g agar + 400 mL distilled water), and morphology was observed and colony counts were calculated. The results were compared with Group I to evaluate the antibacterial effect.

[0044] like Figure 5 As shown in (a), compared with the control group, the m-RuV-COF group showed almost no change in Staphylococcus aureus, while the m-RuV-COF + H2O2 + laser group showed better bactericidal effect. Figure 5(b) It can be seen that the survival rate of Staphylococcus aureus in the m-RuV-COF + laser group decreased to 29.15±1.86%, and the survival rate of Staphylococcus aureus in the m-RuV-COF + H2O2 group also decreased to 18.7±1.05%, indicating that m-RuV-COF can catalyze the decomposition of low-concentration H2O2 into toxic substances. . OH (hydroxyl radical) kills bacteria, and laser irradiation further enhances the sterilization efficiency, almost eliminating all Staphylococcus aureus. The survival rate of Staphylococcus aureus in the m-RuV-COF+H2O2+ laser group was 3.25±0.25%. However, even under various conditions, the m-RuV-COF+H2O2+ laser group did not show good sterilization effects on Escherichia coli, with a survival rate of 70.08±1.56%. The p-RuV-COF+H2O2+ laser group showed no significant sterilization effect on either Staphylococcus aureus or Escherichia coli.

[0045] To further investigate antibacterial properties, the ability to resist biofilm formation was precisely estimated using crystal violet staining. The cells were divided into five groups: (I) PBS, (II) m-RuV-COF, (III) m-RuV-COF + laser, (IV) m-RuV-COF + H2O2, and (V) m-RuV-COF + H2O2 + laser. Figure 5 As shown in (c), group V of the m-RuV-COF+H2O2+ laser group exhibited the strongest biofilm disruption, eliminating 69.19%±2.45% of the biofilm at a sample dose of 125 μg / mL, significantly higher than other groups. These results strongly validate that m-RuV-COF can also serve as a promising anti-biofilm agent.

[0046] Experimental Example 2: Transmission Electron Microscopy Observation of Bacterial Morphology and Staining Tests To further understand the aforementioned antibacterial effects, TEM was used to study the morphological changes of *Escherichia coli* and *Staphylococcus aureus*. First, *Staphylococcus aureus* and *Escherichia coli* were divided into three groups: (I) PBS, (II) m-RuV-COF+ laser, and (III) m-RuV-COF+H2O2+ laser group, where the m-RuV-COF concentration was 125 μg / mL and the laser power was 1.5 W / cm². 2(638 nm, 10 min), H2O2 concentration 10 mmol / mL, PBS neutral. Each group of solutions was fixed in 2.5 wt% glutaraldehyde solution at 4 °C for 24 h, then washed three times with PBS, embedded in agar and blocked. Bacteria were dehydrated by sequentially treating them with ethanol solutions of 30 wt%, 50 wt%, 70 wt%, 90 wt%, 95 wt%, and 100 wt% concentrations for 10 min, followed by acetone treatment for 3 h, gradient permeation embedding. Finally, the bacteria were negatively stained, fixed on a nickel grid, and observed under a transmission electron microscope.

[0047] like Figure 6 As shown in (a), regardless of the laser irradiation intensity, the *E. coli* bacteria treated in each group maintained their structural integrity and had clearly visible flagella, indicating that at a concentration of 125 μg / mL, m-RuV-COF had minimal inhibitory effect on *E. coli*. Conversely, when observing *Staphylococcus aureus*, only the *Staphylococcus aureus* in the PBS group had a smooth and rounded surface. The surface of *Staphylococcus aureus* in the m-RuV-COF+ laser group also showed significant changes, with cell integrity being disrupted, although some bacteria still survived. A large number of *Staphylococcus aureus* bacteria were clearly observed to have died in the m-RuV-COF+H2O2+ laser group.

[0048] Take 400 μL of solution from each group and add 20 μL of SYTO-9 (1.0 × 10⁻⁶) to it. -3 M) and 20 μL PI (1.5 × 10 -3 The solution was incubated in the dark at 37°C for 15 min. Afterwards, excess SYTO-9 and PI were removed by centrifugation. The bacteria in each solution were then resuspended in 50 μL of PBS, and images were observed using an inverted fluorescence microscope.

[0049] like Figure 6 As shown in (b), all Staphylococcus aureus bacteria in the PBS group survived and showed green fluorescence. After treatment with the m-RuV-COF+ laser group, significant red fluorescence was detected, indicating that a large number of bacteria had died. The bacteria treated with m-RuV-COF+ H2O2+ laser showed the strongest red fluorescence, indicating a significant increase in the number of dead bacteria after treatment. All groups of Escherichia coli showed green fluorescence; only the two laser-irradiated groups showed weak red fluorescence, indicating that the treatment had minimal inhibitory effect on E. coli.

[0050] Experimental Example 3: In vitro biocompatibility experiment (1) Cell hemolysis experiment Blood was collected from 5-week-old female KM mice (purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd.). Fresh blood was centrifuged (10000 rpm, 20 min), and the supernatant was discarded to collect red blood cells. The cells were then washed with PBS buffer at a ratio of approximately 1:1 (PBS buffer to red blood cells). After centrifugation, the supernatant was discarded, and this process was repeated four times. Red blood cells were then mixed with PBS buffer at a volume ratio of 3:11. The resulting mixture was then combined with m-RuV-COF (concentrations of 50, 75, 100, 125, and 150 μg / mL) at a ratio of 1:9 (v / v) and incubated at 37°C for 4 h. 100 μL of the supernatant from each tube was added to a 96-well plate, and the absorbance was measured using a microplate reader. Distilled water served as a positive control, and the PBS group served as a negative control. The calculation formula is as follows: Hemolysis volume (%) = (A-An) / (Ap-An) × 100%; A: The absorbance obtained by taking the supernatant after adding M-RUV-COF to red blood cells; An: Absorbance obtained by taking the supernatant after adding PBS to red blood cells (negative control). Ap: Absorbance obtained by taking the supernatant after adding distilled water to red blood cells (positive control).

[0051] like Figure 7 As shown in (a), after co-culturing with blood, m-RuV-COF exhibited concentration-dependent hemolytic behavior, with the hemolysis rate increasing with increasing concentration. Within the experimental concentration range (50, 75, 100, 125, 150 μg / mL), the hemolysis rate remained below the standard value (4.0%), indicating that m-RuV-COF has good blood compatibility.

[0052] (2) Cytotoxicity test In 96-well plates, mouse 3T3 fibroblasts (from the Cell Bank of the Chinese Academy of Sciences) were cultured at a density of 5 × 10⁶ cells per well. 3 Cells were seeded at a density of 100 μL per well, and the edges of the plate were sealed with 100 μL of PBS to prevent excessive evaporation. After 24 h of incubation, different concentrations of m-RuV-COF (0, 50, 75, 100, 125, and 150 μg / mL) were added. After standing for 24 h, the supernatant was discarded, and 10 μL of MTT (tetramethylazazole blue, 5 mg / mL) was added to each well. After incubation for 4 h, the supernatant was aspirated, and 100 μL of dimethyl sulfoxide was added. After standing for approximately 10 min, the absorbance was measured at 540 nm using a microplate reader. Each experiment was repeated three times.

[0053] like Figure 7 As shown in (b), at a high concentration (150 μg / mL) -1Under these conditions, the survival rate of 3T3 cells treated with m-RuV-COF was still 79.3%, indicating that m-RuV-COF has excellent cell compatibility.

[0054] (3) Cell scratch test Prepare a 6-well plate and seed mouse 3T3 fibroblasts (from the Cell Bank of the Chinese Academy of Sciences) into the 6-well plate and culture for 24 h. Using the tip of a 20 μL sterile pipette, gently and slowly scrape the center of the 3T3 cell monolayer to divide the 3T3 cells in the 6-well plate into two halves. After washing with autoclaved PBS to remove detached cells, add 2 mL of fresh DMEM basal medium containing 0, 50, 75, 100, 125, and 150 μg / mL m-RuV-COF, respectively. Microscopic images were taken at 0 h, 12 h, 24 h, and 48 h for comparison. The distance of cell migration was measured using ImageJ software, and the scratch healing rate was calculated using the formula: (scratch area at 0 h - scratch area at different times) / scratch area at 0 h, thus assessing the effect of the material on cell migration ability.

[0055] like Figure 7 (c) and Figure 7 As shown in (d), compared with the control group, m-RuV-COF in each group did not affect cell migration ability. Moreover, within a certain concentration range, it could significantly promote cell migration and accelerate wound healing. This result indicates that m-RuV-COF has almost no effect on the migration and invasion ability of normal cells.

[0056] Experiment Example 4: Wound Healing Experiment Wound model was established using 5-week-old female KM mice weighing approximately 30g (purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd.). Mice were randomly divided into 6 groups: (I) blank, (II) PBS, (III) m-RuV-COF, (IV) m-RuV-COF + laser, (V) m-RuV-COF + H2O2, and (VI) m-RuV-COF + H2O2 + laser, with 6 mice in each group. Normally fed mice served as the blank group (I) for comparison with the other 5 groups. Before formal infection, the weight of each group of mice was measured. Except for the blank group, all other groups of mice were anesthetized, their back hair was shaved, and the wounds were disinfected with 75wt% ethanol to create a wound with a radius of approximately 5mm. Staphylococcus aureus (1×10⁻⁶) was added to the wound. 6 (CFU / mL) After 24 hours of infection, mice in each group were treated according to their assigned groups. The treatment involved adding 0.5 mL of m-RuV-COF to the wound site in each group. The concentration of m-RuV-COF was 125 μg / mL, and the concentration of H2O2 was 10 mmol / mL. The laser irradiation power was 1.5 W / cm².-2 The irradiation time was 10 min. Changes in the back wounds of mice were recorded on days 1, 3, 5, 7, and 9, and the mice's weight was monitored daily. The weight changes and back wound area were plotted. On day 9, mice were anesthetized, and blood was collected from the eyeballs. After blood collection, the mice were euthanized, and the back wounds and major organ tissues (heart, liver, spleen, lungs, and kidneys) were fixed with 10% formalin. Then, H&E staining and Masson's trichrome staining were performed. Two mL of blood was collected from each group for complete blood count analysis.

[0057] Figure 8 (a) and Figure 8 (c) shows the wound area of ​​mice under different treatments on days 1, 3, 5, 7, and 9. On day 1, significant wound exudate was observed in all groups, indicating successful establishment of the animal model. With increasing time, the wound area in all groups decreased significantly. Compared to the PBS and m-RuV-COF groups, the wound repair process in the other three groups was significantly accelerated. Notably, the m-RuV-COF + H2O2 + laser group showed the best wound healing. Furthermore, as... Figure 8 As shown in (b), no significant changes in body weight were detected; body weight increased slowly throughout the treatment period.

[0058] Histological analysis of the skin wounds of the mice in the above groups was performed using hematoxylin-eosin (H&E) staining and Masson staining. Figure 9 As shown, regeneration of intact epidermal tissue and skin structures (such as capillaries), as well as the presence of continuous collagen fibers (stained blue), were observed in the m-RuV-COF+ laser-treated group, and the wounds were almost completely closed. These results indicate that m-RuV-COF+H2O2+ laser treatment significantly promotes wound healing. Furthermore, Figure 10 H&E staining of the heart, liver, spleen, lungs and kidneys of the mice in the above groups showed that no abnormal lesions, inflammatory manifestations or histological changes were observed in any of the major organs of the mice in the above groups, indicating that m-RuV-COF has good biocompatibility in vivo and has no significant effect on tissues and organs.

[0059] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A covalent organic framework polymer with selective inactivation of Staphylococcus aureus, characterized in that, The covalent organic framework polymer is obtained by polymerizing a ruthenium complex and a heteropolyacid via an acetal-amine reaction. The ruthenium complex is bis(hexafluorophosphate)tris(5,5'-bis(diethoxymethyl)-2,2'-bipyridine)ruthenium; the heteropolyacid is Tris-V6O. 19 .

2. The covalent organic framework polymer according to claim 1, characterized in that, The covalent organic framework polymer is prepared by the following method: bis(hexafluorophosphate)tris(5,5'-bis(diethoxymethyl)-2,2'-bipyridine)ruthenium and Tris-V6O 19 A solvothermal reaction was carried out by adding an organic solvent to obtain a covalent organic framework polymer that selectively inactivates Staphylococcus aureus.

3. The covalent organic framework polymer according to claim 2, characterized in that, The bis(hexafluorophosphate)tris(5,5'-bis(diethoxymethyl)-2,2'-bipyridine)ruthenium and Tris-V6O 19 The molar ratio is 1:

2.

4. The covalent organic framework polymer according to claim 2, characterized in that, The organic solvent is obtained by mixing trimethylbenzene, 1,4-dioxane and glacial acetic acid in a volume ratio of 2:2:0.

8.

5. The covalent organic framework polymer according to claim 2, characterized in that, The solvothermal reaction was carried out at a temperature of 180°C for 72 hours.

6. The covalent organic framework polymer according to claim 1, characterized in that, The bis(hexafluorophosphate)tris(5,5'-bis(diethoxymethyl)-2,2'-bipyridine)ruthenium alloy was prepared by the following method: 2,2'-bipyridine-5,5'-dicarboxaldehyde and ruthenium trichloride trihydrate were added to ethanol under a protective atmosphere and refluxed. A saturated ammonium hexafluorophosphate solution was added, and the mixture was filtered and washed to obtain an orange-red solid, namely bis(hexafluorophosphate)tris(5,5'-bis(diethoxymethyl)-2,2'-bipyridine)ruthenium.

7. The covalent organic framework polymer according to claim 6, characterized in that, The molar ratio of 2,2'-bipyridine-5,5'-dicarboxaldehyde to ruthenium trichloride trihydrate is 3.1:

1.

8. The covalent organic framework polymer according to claim 6, characterized in that, The reflux reaction was carried out at a temperature of 80°C for 72 hours.

9. The use of the covalent organic framework polymer according to any one of claims 1 to 8 in the preparation of a drug for selectively inactivating Gram-positive bacteria, characterized in that, The Gram-positive bacteria is Staphylococcus aureus.

10. The application according to claim 9, characterized in that, When the drug concentration was 125 μg / mL, the survival rate of Staphylococcus aureus was 3.25%, and the survival rate of Escherichia coli was 70.08%.

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