High positive charge self-assembled ruthenium complex targeting interference electron transport chain and preparation method and application thereof

Abstract

The application provides a high positive charge self-assembled ruthenium complex targeting electron transport chain and a preparation method and application thereof, and belongs to the technical field of biological medicines.The ruthenium complex provided by the application has a structural formula I shown in formula I; wherein 1<=n<=15.The high positive charge self-assembled ruthenium complex targeting electron transport chain is synthesized for the first time, the complex shows high-efficiency anti-MRSA activity, and can eliminate bacteria without toxic side effects, thereby showing good high-efficiency low-toxicity antibacterial activity, and providing a novel high-efficiency high-biocompatibility antibacterial strategy for solving problems such as antibiotic abuse and antibiotic resistance of bacteria.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a highly positively charged self-assembled ruthenium complex that targets and interferes with the electron transport chain, its preparation method, and its application. Background Technology

[0002] The rapid emergence of bacterial resistance poses a significant challenge to public health. Methicillin-resistant Staphylococcus aureus (MRSA) infection is the superbug causing the highest increase in deaths from drug-resistant infections. Furthermore, MRSA infection can cause a variety of diseases, such as bacterial pneumonia, infective endocarditis, skin and soft tissue infections, deep tissue infections, bacteremia, and orthopedic implant infections. It has a high morbidity and mortality rate, and bacteria are beginning to develop resistance to many commonly used clinical drugs. Bacteria typically develop resistance through various means, such as reducing drug permeability and increasing antibiotic efflux, modifying or altering the bacterial antibiotic target, and modifying or destroying the drug itself to inactivate it. Bacteria can also form biofilms, which firmly adhere to contact surfaces, encapsulating bacterial cells in a three-dimensional gel structure. This structure resists both external factors (such as antibiotics and ultraviolet light) and internal factors (such as host immune cell drugs and blocking immune cell activation). These behaviors render existing antibiotics ineffective. Traditional antibacterial drugs generally work by interfering with cell wall synthesis, disrupting bacterial cell membranes, interfering with bacterial nucleic acid synthesis, and interfering with bacterial protein synthesis. With the rapid development of drug-resistant bacteria, traditional antibiotics are no longer effective against new superbugs. Therefore, there is an urgent need to develop new antibacterial mechanisms to circumvent bacterial resistance and achieve highly effective antibacterial results.

[0003] Metal-based antibacterial agents are considered a promising solution to the current antibiotic crisis due to their unique three-dimensional geometry and physical properties, as well as multiple mechanisms of action. The mechanisms of action of metal-based antibiotics include β-lactamase inhibitors, metalloproteinase inhibitors, reactive oxygen species inducers, etc. In addition to the above mechanisms, disrupting bacterial electron transport is a promising complementary strategy that can enhance bactericidal efficacy. Electron transport is a crucial physiological process in bacteria, supporting basic functions such as respiration, energy production, and growth. Bacteria generate the energy needed for survival and proliferation through an electron transport chain embedded in the cell membrane. Disrupting this process impairs their metabolic functions, leading to energy depletion and ultimately cell death. Currently, antibacterial drugs that act on the bacterial electron transport chain (ETC) aim to interfere with this key energy metabolic process of transmembrane electron transport. The bacterial ETC is central to its oxidative phosphorylation system, responsible for establishing a proton gradient in the stepwise transfer of electrons to the final receptor, thereby driving ATP synthesis. Many proteins in the bacterial ETC are responsible for electron transport, and these can all be targets. Currently, there are relatively few drugs that exert antibacterial effects by affecting the ETC in other ways. Therefore, interfering with bacterial ETC is a promising antibacterial mechanism. Summary of the Invention

[0004] Based on the above, the present invention aims to provide a highly positively charged self-assembled ruthenium complex that targets and interferes with the electron transport chain, its preparation method, and its application. This invention is the first to synthesize a highly positively charged self-assembled ruthenium complex that targets the electron transport chain. This complex exhibits highly efficient anti-MRSA activity and, while capable of eliminating bacteria, has no toxic side effects, demonstrating excellent, highly efficient, and low-toxicity antibacterial activity.

[0005] To achieve the above objectives, the present invention provides the following technical solution: One of the technical solutions of this invention is a highly positively charged self-assembled ruthenium complex Ru1 that targets and interferes with the electron transport chain, having the structure shown in Formula I: Formula I; Where 1≤n≤15.

[0006] The second technical solution of the present invention is a method for preparing the above-mentioned highly positively charged self-assembled ruthenium complex that targets and interferes with the electron transport chain, comprising the following steps: 2-2-(pyridine)benzimidazole was reacted with compound 1 under alkaline conditions in organic solvent 1 to obtain ligand 1; 4,4'-bis(bromomethyl)-2,2'-bipyridine and trimethylamine were reacted in organic solvent 2 to obtain ligand 2; Under a protective atmosphere, cis-[RuCl2(DMSO)4] and ligand 1 undergo a coordination substitution reaction in a mixed solvent to obtain a ruthenium precursor complex containing ligand 1; The ruthenium precursor complex containing ligand 1 and ligand 2 undergo a ligand exchange reaction to obtain the highly positively charged self-assembled ruthenium complex. The structural formula of compound 1 is ; The structural formula of ligand 1 is ; The structural formula of ligand 2 is ; The structural formula of the ruthenium precursor complex containing ligand 1 is as follows: ; In compound 1, ligand 1, and the ruthenium precursor complex containing ligand 1, n is consistent with the n in the structural formula shown in Formula I.

[0007] The third technical solution of the present invention is the application of the above-mentioned highly positively charged self-assembled ruthenium complex that targets and interferes with the electron transport chain in the preparation of drugs for the prevention and treatment of diseases caused by bacterial infections.

[0008] The fourth technical solution of the present invention is a drug for preventing and treating diseases caused by bacterial infection, comprising the above-mentioned highly positively charged self-assembled ruthenium complex that targets and interferes with the electron transport chain.

[0009] The present invention discloses the following technical effects: This invention synthesizes a highly positively charged self-assembled ruthenium complex and tests its activity against drug-resistant bacterial infections. In vitro antibacterial and cytotoxicity experiments verified the high efficiency and good biocompatibility of the highly positively charged self-assembled ruthenium complex against MRSA. Furthermore, this ruthenium complex can target bacterial peptidoglycan, affecting intracellular NAD+ in bacteria. + The NADH balance disrupts the bacterial electron transport chain, causing an imbalance in the bacterial redox pathway, leading to the accumulation of reactive oxygen species and a decrease in ATP content, resulting in energy metabolism breakdown. This demonstrates excellent, highly effective, and low-toxicity antibacterial activity. In summary, this invention provides a novel, highly efficient, and highly biocompatible antibacterial strategy to address the problems of antibiotic abuse and antibiotic resistance in bacteria. Attached Figure Description

[0010] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0011] Figure 1The NMR spectra of Ru1 obtained in Example 1 of this invention are 1H NMR spectrum a and 1C NMR spectrum b.

[0012] Figure 2 The NMR spectra of Ru2 obtained in Example 1 of this invention are 1H NMR spectrum a and 1C NMR spectrum b.

[0013] Figure 3 This document describes the synthesis and characterization of the product obtained in Example 1 of this invention. Specifically, a represents the synthesis route and self-assembly diagram of Ru1; b represents the verification of the Zeta potential and Tyndall effect of Ru1; c and d represent the critical aggregation concentration (CAC) measurements of Ru1 and Ru2, respectively; e represents the transmission electron microscopy (TEM) imaging of Ru1; f represents the energy-dispersive X-ray spectroscopy (EDS) analysis of Ru1; g and h represent the self-assembled structures of Ru1 and Ru2 obtained from molecular dynamics simulations, respectively; i represents the root mean square deviation (RMSD); j represents the solvent accessible surface area (SASA); and k represents the interaction energy obtained from molecular dynamics simulations of Ru1 and Ru2.

[0014] Figure 4 This document describes the antibacterial activity study of Ru1 in Example 2 of the present invention. Specifically, a) is a photograph of MRSA treated with the Oxford cup method and its quantitative analysis results; c) is a photograph of diluted MRSA plates treated with different concentrations of Ru1 and the corresponding quantitative analysis results of colony counts; e) is the time-dependent inhibition curves of MRSA treated with Ru1 and Ru2 respectively; f) is the drug resistance induction experiment of methicillin, vancomycin, and Ru1 on MRSA; g) is the flow cytometry analysis of MRSA uptake after treatment with different concentrations of Ru1 and its quantitative analysis results; i) is the flow cytometry detection of MRSA uptake of Ru1 and Ru2; j) is the SYTO 9 / PI fluorescence imaging and red-green fluorescence ratio (m) of MRSA after different drug treatments; k) is the membrane potential fluorescence imaging and n quantitative analysis results of MRSA after different drug treatments; and l is the SEM imaging of MRSA after different drug treatments.

[0015] Figure 5 This invention relates to the study of the antibacterial mechanism of Ru1 in Example 3; wherein, a) is TEM image and EDS energy dispersive spectroscopy analysis of MRSA after treatment with Ru1 and Ru2, b) is the targeting competitive binding experiment of peptidoglycan and c) teichoic acid, d) is the binding ability of Ru1 to teichoic acid determined by ITC method, e) is the electron transport chain activity of MRSA after treatment with Ru1 and Ru2 determined by INT method and f) is the quantitative analysis result, and g NAD of MRSA after treatment with Ru1 and Ru2 is... + Changes in the NADH ratio and hATP levels.

[0016] Figure 6This is an in vivo anti-infection study of Ru1 in Implementation Example 4 of the present invention; wherein, a is a schematic diagram of the experimental procedure of the mouse wound infection model, b is a photo of diluted plating of bacteria from mouse wounds on day 11 after different drug treatments and colony count analysis, c is the morphology of microneedles and the puncture effect of agarose to simulate skin, d is the changes in mouse wounds within 11 days after different drug treatments, and e is HE staining, f is Masson staining, and g is Giemsa staining of the skin at the mouse wound site on day 11. h, i, and j are the body weight, temperature, and wound healing rate of mice within 11 days after different drug treatments, respectively.

[0017] Figure 7 This is an in vivo anti-infection study of Ru1 in Implementation Example 4 of the present invention; wherein, a is a schematic diagram of the experimental procedure of the mouse systemic infection model, b is a photo of the diluted plating of the main organs of the mouse after different drug treatments and the colony count analysis of each organ c, d, e, and f, and g is a histological section analysis of the main organs of the mouse after different drug treatments.

[0018] Figure 8 This is a biosafety evaluation of Ru1 in Implementation Example 5 of the present invention; wherein, a, b, and c are the cell viability test results of three normal mammalian cells under different concentrations of Ru1, d is the hemolysis of fresh red blood cells after treatment with different concentrations of Ru1, and e is the analysis of the complete blood count results of mice after treatment with different drugs. Detailed Implementation

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included within the scope of this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0021] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0022] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0023] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0024] The first aspect of this invention provides a highly positively charged self-assembled ruthenium complex that targets and interferes with the electron transport chain, having the structure shown in Formula I: Formula I; Where 1≤n≤15.

[0025] In a preferred embodiment of the present invention, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0026] A second aspect of the present invention provides a method for preparing the above-mentioned highly positively charged self-assembled ruthenium complex that targets and interferes with the electron transport chain, comprising the following steps: 2-2-(pyridine)benzimidazole was reacted with compound 1 under alkaline conditions in organic solvent 1 to obtain ligand 1; 4,4'-bis(bromomethyl)-2,2'-bipyridine and trimethylamine were reacted in organic solvent 2 to obtain ligand 2; Under a protective atmosphere, cis -[RuCl2(DMSO)4] and ligand 1 undergo a coordination substitution reaction in a mixed solvent to obtain a ruthenium precursor complex containing ligand 1; The ruthenium precursor complex containing ligand 1 and ligand 2 undergo a ligand exchange reaction to obtain the highly positively charged self-assembled ruthenium complex. The structural formula of compound 1 is ; The structural formula of ligand 1 is ; The structural formula of ligand 2 is ; The structural formula of the ruthenium precursor complex containing ligand 1 is as follows: ; In compound 1, ligand 1, and the ruthenium precursor complex containing ligand 1, n is consistent with the n in the structural formula shown in Formula I.

[0027] In a preferred embodiment of the present invention, the molar ratio of 2-2-(pyridine)benzimidazole to compound 1 is 1:1 to 1:3; the reagent providing the base in the alkaline conditions is potassium carbonate; the organic solvent 1 is N,N-dimethylformamide (DMF); the temperature of reaction 1 is 50 to 90°C, and the time is 6 to 10 hours.

[0028] In a preferred embodiment of the present invention, the molar ratio of 2-2-(pyridine)benzimidazole to potassium carbonate is 1:1 to 1:3.

[0029] In a preferred embodiment of the present invention, after reaction 1 is completed, the reaction solution is further diluted with water, then extracted with ethyl acetate, then extracted with saturated brine to obtain an organic layer, and then purified by passing the organic layer through a 200-300 mesh silica gel column using dichloromethane and ethyl acetate as developing solvents (the volume ratio of dichloromethane to ethyl acetate is 20:1).

[0030] The present invention does not impose any special limitation on the amount of organic solvent 1 used, as long as the amount is sufficient to fully dissolve the raw materials and satisfy the reaction 1 to proceed.

[0031] In a preferred embodiment of the present invention, the molar ratio of 4,4'-bis(bromomethyl)-2,2'-bipyridine to trimethylamine is 1:8 to 1:12; the organic solvent 2 is acetonitrile; the reaction 2 is carried out by stirring at room temperature for 0.5 to 2 hours, followed by rotary evaporation.

[0032] The present invention does not impose any special limitation on the amount of organic solvent 2 used, as long as the amount is sufficient to fully dissolve the raw materials and satisfy the reaction 2 to proceed.

[0033] In a preferred embodiment of the present invention, the molar ratio of cis-[RuCl2(DMSO)4] to ligand 1 is 1:1 to 1:3; the mixed solvent is a mixture of ethanol and dimethyl sulfoxide (DMSO); the temperature of the coordination substitution reaction is 70 to 100°C, and the time is 3 to 6 h.

[0034] In a preferred embodiment of the present invention, the volume ratio of ethanol to DMSO is 16:1. The present invention does not impose any special limitation on the amount of the mixed solvent, as long as it is sufficient to fully dissolve the raw materials and satisfy the requirements for the coordination substitution reaction.

[0035] In this invention, after the coordination substitution reaction is completed, the resulting reaction solution is rotary evaporated, and then ligand 2 is added to the rotary evaporated reaction solution to carry out a ligand exchange reaction.

[0036] In a preferred embodiment of the present invention, the molar ratio of ligand 1 to ligand 2 is 1:1 to 1:3; the temperature of the ligand exchange reaction is 110 to 150°C, and the time is 8 to 13 hours.

[0037] In a preferred embodiment of the present invention, the solvent used for the ligand exchange reaction is a mixed solvent of ethanol and water, with a volume ratio of ethanol to water of 1:1. The present invention does not impose any special limitation on the amount of the mixed solvent of ethanol and water, as long as the amount is sufficient to fully dissolve the raw materials and allow the ligand exchange reaction to proceed.

[0038] In a preferred embodiment of the present invention, after the ligand exchange reaction is completed, the process further includes purifying the solution by passing it through a 300-400 mesh alumina column using dichloromethane and methanol as the developing solvent (the volume ratio of dichloromethane to methanol is 5:1), collecting the red solution, and then evaporating it by rotary evaporation.

[0039] A third aspect of the present invention provides the application of the above-mentioned highly positively charged self-assembled ruthenium complex that targets and interferes with the electron transport chain in the preparation of medicaments for the prevention and treatment of diseases caused by bacterial infections.

[0040] In a preferred embodiment of the present invention, the disease caused by bacterial infection is a disease caused by methicillin-resistant Staphylococcus aureus infection.

[0041] A fourth aspect of the present invention provides a medicament for the prevention and treatment of diseases caused by bacterial infection, comprising the above-mentioned highly positively charged self-assembled ruthenium complex that targets and interferes with the electron transport chain.

[0042] In a preferred embodiment of the present invention, the disease caused by bacterial infection is a disease caused by methicillin-resistant Staphylococcus aureus infection.

[0043] The medicaments for preventing and treating diseases caused by bacterial infections in this invention also include pharmaceutically acceptable excipients.

[0044] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0045] To better understand the present invention, the following embodiments further illustrate the content of the present invention, but the content of the present invention is not limited to the following embodiments.

[0046] Example 1 Synthesis and Characterization (1) Synthetic ligand 1 (n=15): 1-Octadecyl-2-2-(pyridine)benzimidazole 2,2-(pyridine)benzimidazole (MW: 195.23, 1 mmol) and K2CO3 (MW: 138.206, 2 mmol) were placed in a round-bottom flask, dissolved in 3 mL of DMF, and stirred at 70 °C for 30 min. Then, bromooctadecane (MW: 333, 1.5 mmol) was added, and the mixture was reacted at 70 °C for 8 h. After the reaction was completed, the reaction solution was diluted with water, extracted with ethyl acetate, and then extracted with saturated brine to obtain an organic layer. The organic layer was then purified by passing the solution through a 200-300 mesh silica gel column using dichloromethane and ethyl acetate as the developing solvent (dichloromethane to ethyl acetate volume ratio of 20:1), and evaporated to dryness to obtain a white solid. (2) Synthesis of ligand 2: 4,4'-bis(trimethylamine)-2,2'-bipyridine Take 0.1 mmol of 4,4'-bis(bromomethyl)-2,2'-bipyridine, 1 mmol of trimethylamine and 10 mL of anhydrous acetonitrile in a reaction tube, stir at room temperature for 1.5 h, and then evaporate to dryness to obtain the final product.

[0047] (3) Synthesis of highly positively charged self-assembled ruthenium complexes (Ru1) that target and interfere with the electron transport chain Pick cis [RuCl2(DMSO)4](DMSO-O) (0.2 mmol) and 1-octadecyl-2-2-(pyridine)benzimidazole (0.21 mmol) were dissolved in a round-bottom flask with 16 mL of ethanol and 1 mL of DMSO. The mixture was reacted at 80 °C for 4 h under argon protection. After the reaction was complete, the resulting reaction solution was rotary evaporated. The remaining reaction solution was then transferred to a high-pressure reactor, where 4,4'-bis(trimethylamine)-2,2'-bipyridine (MW: 460, 0.39 mmol), 6 mL of ethanol, and 6 mL of water were added. The mixture was reacted in an oil bath at 150 °C for 12 h. After the reaction was complete, the solution was purified by passing it through a 300-400 mesh alumina column using dichloromethane and methanol as the developing solvent (dichloromethane to methanol volume ratio of 5:1). The red solution was collected and evaporated to dryness to obtain the solid, which is Ru1 (n=15).

[0048] By replacing bromooctadecane with 1-bromobutane, and keeping the other steps and parameters the same as those for synthesizing Ru1, Ru2 (n=1) was prepared.

[0049] Comprehensive application of nuclear magnetic resonance (NMR) 1 H-NMR, 13 The structure of the synthesized complex was analyzed by methods such as C-NMR, UV-Vis absorption spectroscopy, fluorescence, particle size potential, TEM, elemental analysis, and HPLC.

[0050] Depend on Figure 1 , Figure 2 , Figure 3It is evident that Ru1 and Ru2 were successfully synthesized.

[0051] Example 2 Antibacterial activity study (1) Minimum inhibitory concentration (MIC) The minimum inhibitory concentrations (MICs) of different treatments against different bacterial strains were determined using the clinical laboratory standard research broth microdilution method. First, bacteria were incubated overnight in LB medium at 37°C on a shaker (180–200 rpm) until the logarithmic growth phase was reached. The precipitate was then collected by centrifugation and washed three times with 0.85% sodium chloride solution. The concentration of the suspended bacteria was determined by the optical density (OD 600) measured at 600 nm. The bacterial solution was diluted with culture medium to a final concentration of 1 × 10⁻⁶. 6 CFU / mL. 100 µL of these solutions were added to 96-well plates containing different concentrations of the drug (100 µL) to achieve a final bacterial concentration of approximately 5 × 10⁻⁶. 5 CFU / mL. Bacteria were cultured at 37°C for 24 h, and the OD600 value of the bacterial suspension was measured using a multi-functional microplate reader to calculate the minimum inhibitory concentration (MIC). The MIC was determined as the treatment concentration at which no microbial growth was observed. Each test was repeated three times.

[0052] (2) Oxford cup inhibition zone determination Different drugs were mixed with MRSA at a volume ratio of 1:1 to achieve a final concentration of 4 μM for each drug and a final concentration of 1 × 10⁻⁶ for MRSA. 6 CFU / mL, place Oxford cups in petri dishes, add 10 μL of the above mixed solution to each Oxford cup, and then place the petri dishes in a 37°C incubator for 24 h. Use PBS-treated bacteria as a control and photograph and record the inhibition zone.

[0053] (3) Diluted coating plate method Add Ru1 to a concentration of 5×10 5 In CFU / mL MRSA bacterial suspension, Ru1 concentrations of 4 μM, 2 μM, and 1 μM were prepared, mixed thoroughly, and incubated at 37°C for 24 h. Subsequently, 50 μL of the above mixed solution was added dropwise to each culture dish, spread evenly with a spreader, and incubated at 37°C for 24 h. Bacteria treated with PBS were used as a control to observe colony growth, and the colonies were photographed, recorded, and counted.

[0054] (4) Time-based inhibition curve Add Ru1 and Ru2 to a concentration of 5 × 10⁻⁶. 5 In MRSA bacterial culture at CFU / mL, the final concentrations of Ru1 and Ru2 were adjusted to 4 μM. OD values ​​were measured every 1 hour from 0 h to 12 h. 600The value was used as a control for bacteria treated with PBS to observe the drug's ability to inhibit bacterial growth within 12 hours.

[0055] (5) Bacterial resistance induction experiment 5×10 5 CFU / mL MRSA bacterial suspensions were co-incubated with different concentrations of Ru1, vancomycin, and methicillin. After 24 hours of incubation, the MIC was determined according to the method described above. Bacterial suspensions were taken from the wells containing the highest drug concentration that enabled bacterial growth and incubated with fresh LB medium, followed by co-incubation with each test drug. This process was repeated for 20 days, and the fold change in the MIC of the bacteria for each drug was observed.

[0056] (6) Staining of live and dead bacteria Add 1 mL of each drug to a concentration of 3×10⁻⁶. 8 The final concentrations of each drug were adjusted to 4 μM by adding CFU / mL MRSA bacterial suspensions and incubating at 37°C for 3 h. The bacterial pellet was then centrifuged, washed three times with PBS, stained with SYTO9 / PI, and incubated at 37°C in the dark for 30 min, followed by three washes with PBS. The antibacterial effects of each drug were observed using a laser confocal microscope (IXplore SpinSR, Olympus, Japan), with PBS-treated bacteria serving as a control.

[0057] (7) Measurement of bacterial membrane potential Add 1 mL of each drug to a concentration of 3×10⁻⁶. 8 The final concentration of each drug was 4 μM in CFU / mL MRSA bacterial culture, and the mixture was incubated at 37°C for 3 h. 10 mM DiBAC4(3) was diluted with DMSO to prepare a 1 mM working solution. Then, 5 μL of DiBAC4(3) working solution was added to each bacterial mixture, and the mixture was incubated at 37°C in the dark for 20 min. The images were taken using a laser confocal microscope, with PBS-treated bacteria as a control, to observe the effect of each drug on the membrane potential.

[0058] (8) Bacterial uptake experiment Different drug groups: Ru1 and Ru2 were added to 1 mL of a concentration of 3×10⁻⁶. 8 The final concentration of each drug was 4 μM in CFU / mL MRSA bacterial culture, and the culture was incubated at 37°C for 3 h. The bacteria were washed three times with PBS and the fluorescence intensity was measured by flow cytometry. The PBS-treated bacteria were used as a control group to observe the changes in bacterial fluorescence intensity.

[0059] Different concentration groups: Ru1 was added to 1 mL of solution with a concentration of 3×10⁻⁶. 8In CFU / mL MRSA bacterial suspension, Ru1 concentrations of 2 μM, 4 μM, 8 μM, and 16 μM were added and incubated at 37°C for 3 h. The suspension was washed three times with PBS and analyzed by flow cytometry. Bacteria treated with PBS were used as a control group to observe changes in bacterial fluorescence intensity.

[0060] Depend on Figure 4 It is evident that Ru exhibits good antibacterial effects. Using the Oxford cup method to detect inhibition zones, the inhibition zone of Ru1 was significantly smaller than that of Ru2 and the methicillin-treated group, and also slightly smaller than that of the vancomycin group. Figure 4 (a, c). The dilution plating method showed that the antibacterial activity of Ru1 was concentration-dependent, with 4 μM (MIC) being its minimum bactericidal concentration. Figure 4 (b, d). After SYTO9 / PI fluorescent staining, the Ru1 treatment group showed a large amount of red dead bacterial fluorescence, while the Ru2 group showed almost no fluorescence, indicating that the antibacterial effect of Ru1 was superior to that of Ru2. Figure 4 After 20 days of continuous induction of drug resistance, the MIC of MRSA against methicillin increased by 1024 times, against vancomycin by 8 times, and against Ru1 by only 4 times, indicating that Ru1 has better resistance to drug resistance than vancomycin. Figure 4 (f). Flow cytometry analysis showed that Ru1 treatment increased bacterial fluorescence signal intensity in a concentration-dependent manner, while Ru2 treatment did not, indicating that MRSA uptakes Ru1 but not Ru2 (f). Figure 4 (g–i). The membrane potential probe DiBCA4(3) detected a significant change in membrane potential in the Ru1 treatment group, while other treatment groups showed almost no change. Figure 4 (k, n). Scanning electron microscopy revealed that Ru1 treatment resulted in the adhesion of granular nanoassemblies to the surface of MRSA cells, with no significant shrinkage of the cell membrane; vancomycin treatment caused cell shrinkage; methicillin and Ru2 groups showed no significant morphological changes. Figure 4 (l). These results suggest that Ru1 is taken up by bacteria after electrostatic binding, which may affect electron transfer processes rather than directly damaging the cell membrane.

[0061] Example 3 Research on antibacterial mechanisms (1) Target competition binding experiment Peptidoglycan (PGN) was prepared into a stock solution of 5 mg / mL using PBS. Following the method for determining the MIC, when determining the MIC of Ru1, the PGN stock solution was added to each well of a 96-well plate and diluted sequentially to 0.0156-0.5 mg / mL. The plates were then incubated together, and the changes in their MIC values ​​were observed.

[0062] Peptidoglycan (LTA) was prepared into a stock solution of 5 mg / mL using PBS. Following the method for determining the MIC, when determining the MIC of Ru1, the PGN stock solution was added to each well of a 96-well plate and diluted sequentially to 0.0039-0.125 mg / mL. The plates were then incubated together, and the changes in their MIC values ​​were observed.

[0063] (2) ITC determination The binding of Ru1 to lipoteichoic acid was determined using the ITC method. Titration experiments were performed at 25°C using an isothermal titration calorimeter (NANO ITC, USA). The Ru1 solution concentration was 1 mM, and the lipoteichoic acid (LTA) solution concentration was 0.05 mM, both prepared with 10 mM PBS at pH 7.4 and degassed before the experiment.

[0064] 300 μL of LTA solution (0.05 mM) was added to the sample cell, and 50 μL of Ru1 solution (1 mM) was drawn into the syringe. The titration program was set as follows: 20 drops in total, 2 μL per drop, titration interval of 120 s, and stirring speed of 350 r / min. The experimental group used Ru1 solution to titrate the LTA solution; the control group used Ru1 solution to titrate the blank buffer to subtract the heat of dilution. The test data were fitted based on an Independent model to obtain the corresponding dissociation constant K. d (Combined with constant K) a Parameters such as the reciprocal of the stoichiometry (n), enthalpy change (ΔH), and entropy change (ΔS) are also included.

[0065] (3) Electron transport chain activity determination Prepare a 2 mg / ml iodonitrobenzolium chloride (INT) solution and a 50 mg / ml ascorbic acid solution (dissolved in PBS). Then, dilute the INT solution to concentrations of 0.025, 0.05, 0.1, 0.175, and 0.2 mg / ml. Add 40 μL of ascorbic acid solution to each solution, incubate in the dark for several minutes until the solution turns purple-red, and measure the absorbance at 490 nm to plot a standard curve. Incubate bacteria with Ru1 and Ru2 (4 μM) for 3 h, centrifuge to obtain precipitates, add 2 mg / ml INT PBS solution, incubate at room temperature in the dark for 40 min, observe the color change, centrifuge to remove the supernatant, add 1 ml of methanol, mix thoroughly to extract INTF, centrifuge to obtain a methanol solution, repeat the extraction twice (until the methanol is essentially colorless), and then measure the absorbance spectrum of the methanol solution, observing its absorbance at 490 nm. Electron transport activity can be quantified as a U-value, calculated using the formula U=D. 490 V e / KVt, where D 490 V is the absorbance value at 490 nm. eV represents the volume of the extraction solution (mL), K represents the slope of the standard curve, V represents the volume of the mixed solution (mL), and t represents the incubation time (min).

[0066] (4) Bacterial ATP and NAD + / NADH ratio determination First, bacteria were incubated with Ru1 and Ru2 (4 μM) for 3 h, respectively. After centrifugation, the precipitates were obtained, washed twice with PBS, and then resuspended in 150 μL of lysozyme (4 mg / ml, Beyotime, ST206) and vortexed vigorously for 15 minutes to remove the cell wall. ATP levels and the NAD+ / NADH ratio were then measured. ATP levels in MRSA were detected using an ATP chemiluminescence assay kit (Solepro). The simplified steps are as follows: ATP was extracted using the ATP extraction reagent provided in the kit, heated in a boiling water bath for 10 minutes, and then centrifuged at 10,000 rpm for 10 minutes. The supernatant was collected as the test sample. Standard solutions and samples were added to the wells of the assay plate, and the fluorescence value of each well was measured using a multi-mode microplate reader. A standard curve was plotted based on the fluorescence values ​​of the standards, and the ATP level of the sample was calculated accordingly. NAD+ / NADH levels in MRSA... + / NADH ratio through NAD + The NADH colorimetric assay kit (Beyotime) was used for detection. The simplified steps are as follows: NAD was extracted from MRSA using extraction reagents. + NADH was removed by centrifugation using a 10 KD ultrafiltration tube after cell wall removal. + Degrading enzymes. Following the kit's operating procedures, standards of varying concentrations and untreated samples (for NAD detection) are prepared. + The total amount of NADH and the sample after incubation at 60°C for 30 minutes (only the amount of NADH was measured) were added to microplates. After incubation at 37°C for 30 minutes, the absorbance at 450 nm was measured using a microplate reader. The NAD content in the sample was calculated based on the standard curve. + And the content of NADH, and determine the NAD content of each group. + / NADH ratio.

[0067] Depend on Figure 5 As can be seen, TEM and EDS showed that particles adhered to the surface of the MRSA in the Ru1-treated group, and Ru elemental signals were detected. This phenomenon was not observed in the Ru2 and Control groups, confirming that the surface particles were Ru1 nanoparticles. Figure 5 (a) Competitive inhibition experiments showed that the addition of either LTA or PGN increased the MIC of Ru1; at the same concentration, LTA increased the MIC by 64-fold, while PGN only increased it by about 8-fold, indicating that Ru1 has a stronger binding affinity to LTA, and LTA is the main binding site. Figure 5 (b, c). The dissociation constant K of Ru1 and LTA was measured by ITC.d = 2.16 μM, confirming that the two have high affinity ( Figure 5 (d). INT experiments showed that the absorbance at 490 nm was significantly reduced in the Ru1-treated group, indicating that the electron transport chain activity of MRSA was inhibited. Figure 5 (e,f). After Ru1 treatment, NAD of MRSA + Both the NADH ratio and ATP content decreased significantly. Figure 5 (g, h). This indicates that Ru1 binds to LTA on the cell wall with high affinity, inhibiting the electron transport chain and leading to NAD. + NADH imbalance and ATP synthesis blockage ultimately lead to bacterial death due to energy metabolism collapse.

[0068] Example 4 In vivo anti-infection studies (1) Preparation of microneedles The microneedle molds were purchased from Henan Weina Pentium Biotechnology Co., Ltd., China. First, a 20% (w / v) base solution of PVP:PVA (2:1) was prepared. Ru1 and vancomycin were added to the base solution to achieve a concentration of 60 μg / mL for both Ru1 and vancomycin. 300 μL of the different drug mixtures were poured into the microneedle molds, and vacuum was applied to fill the microneedle tips. After removing excess solution, 1 mL of each drug mixture was added to fill the backing. The molds were dried overnight at room temperature. Finally, the microneedles were peeled off from the molds to obtain Ru1 microneedles and vancomycin microneedles.

[0069] (2) Constructing a mouse systemic infection model The feeding and experiments of mice were strictly conducted in accordance with the approval requirements of the Ethics Committee of CAS Detection Technology Service (Guangzhou) Co., Ltd. (Ethics Review No.: CASTS / OR-T0003-02). Female Balb / c mice (6-8 weeks old) were purchased from Hunan SJA Laboratory Animal Co., Ltd. The mice were housed in a room under light / dark cycling for one week. Healthy female Balb / c mice (6-8 weeks old) were divided into four groups (n=6): PBS group, vancomycin group, Ru1 group, and uninfected group. First, 100 μL of MRSA bacterial suspension (1×10⁻⁶) was prepared. 8 A mouse systemic infection model was established by injecting CFU / mL into the tail vein of mice. Two hours after infection, the mice were administered the drug intravenously (3 mg / kg). Twenty-four hours after treatment, the mice were euthanized, and the liver, spleen, lungs, and kidneys were collected for H&E staining and histological examination. The homogenates were then diluted, plated, and CFU counted.

[0070] (3) Constructing a mouse wound infection model Healthy female Balb / c mice (6-8 weeks old) were randomly divided into four groups (n=5): PBS group, vancomycin group, Ru1 group, and uninfected group. Hair was removed with a depilatory agent, and the mice were anesthetized with isoflurane. A circular wound approximately 10 mm in diameter was created, and 50 μL of 2×10 [unspecified medication] was instilled into the wound. 8 An infection model was established using CFU / mL MRSA. On days 1 and 3, mice were administered 50 μL PBS with vancomycin microneedles and Ru1 microneedles loaded with 2 mg / kg of the drug. Mice were weighed, and their body temperature and wound area were measured and photographed. On day 11, bacteria were collected from the mouse wounds using PBS-moistened swabs, diluted, and plated. CFU of residual bacteria at the wound site was used to calculate bacterial residue. Mice were euthanized after anesthesia, and fresh whole blood was collected to assess in vivo biocompatibility. H&E staining, Masson staining, immunohistochemistry, and immunofluorescence staining were performed on different wound sections.

[0071] Depend on Figure 6 , Figure 7 The mouse wound infection model showed that on day 11, the wounds in the Ru1 group healed almost completely, demonstrating better recovery than the vancomycin group and the Control group. Figure 6 (a, h–j). Colony counts indicate that bacteria in the Ru1 group wound were almost completely eradicated ( Figure 6 (b) H&E staining showed that the Ru1 group had significantly less inflammatory cell infiltration than the Control group and the vancomycin group ( Figure 6 Masson staining showed that Ru1 group had a large number of orderly arranged blue collagen fibers (e); Figure 6 (f); Giemsa staining showed that the Ru1 group had very few purple granules, far fewer than the Control group (f). Figure 6 (g). This indicates that Ru1 can effectively clear bacteria from mouse wounds and promote wound healing. In a mouse systemic infection model, the bacterial load in the liver, spleen, lungs, and kidneys of the Ru1-treated group was significantly lower than that of the Control group (g). Figure 7 (b–f). H&E staining of various organs showed that the morphology of the Ru1 group was similar to that of the uninfected group, while the Control group showed obvious inflammatory infiltration and tissue damage. Figure 7 (g). This indicates that Ru1 can clear bacteria from various organs of mice via intravenous injection.

[0072] Example 5 Biosafety assessment (1) CCK8 Experiment Human umbilical vein endothelial cells (HUVECs) and mouse fibroblasts (L929) were respectively 1×10⁶ cells per well. 4Cells were seeded at a density of 1000 cells per well in 96-well plates. The 96-well plates were then placed in an incubator and incubated at 37°C and 5% CO2 for 12 hours. Next, the supernatant was removed, and different concentrations of nanofiber membrane gradient dilutions were added to the cells, and the cells were incubated for 24 hours. After 24 hours, a solution containing 10% (v / v) CCK-8 was added to the cells. After incubation at 37°C for 30 minutes, the absorbance (OD) value of each well was measured using a microplate reader. Cell viability was calculated according to formula (1). Cell viability (%) = (OD-OD0) / (OD1-OD0) (1); where OD0 is the absorbance of the blank group, OD1 is the absorbance of the control group, and OD is the absorbance of the sample. Each experiment was repeated three times.

[0073] (2) Hemolysis test Commercially purchased 4% fresh red blood cells (RBCs, 5 mL) were centrifuged at 3500 rpm for 5 min and gently washed three times with sterile PBS. Different concentrations of Ru1 (100 µL) were added to 300 µL of RBCs, and the solutions were incubated at 37°C for 4 h. At the end of incubation, the solutions were centrifuged at 3500 rpm for 5 min, and hemolytic activity was measured at 540 nm using a microplate reader. Triton X-100 (0.1% in PBS) capable of completely lysing RBCs was used as a positive control, while PBS was used as a negative control. Hemolysis rate (HR) was calculated using the following formula: Hemolysis rate (%) = (OD) / (RBCs + 0.1%) sample - OD negative control ) / (OD positive control -OD negative control ) ×100%; where, OD sample OD negative control and OD positive control These represent the absorbance of the sample, the negative control, and the positive control, respectively.

[0074] The analysis results are shown below. Figure 8 In the safety evaluation, Ru1 maintained >90% viability in L929, Beas-2B, and HUVEC cells within the range of 1–32 μM. Figure 8 (a–c), hemolysis rate <5% in the range of 4–64 μM ( Figure 8 In the wound infection model, there was no significant difference in blood routine tests between the Ru1-treated group and the uninfected group (d). Figure 8 (e). Conclusion: Ru1 can effectively clear bacteria from multiple organs in vivo, protect organs from damage, and has good biosafety within the tested concentration range.

[0075] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A highly positively charged self-assembled ruthenium complex that targets and interferes with the electron transport chain, characterized in that, It has the structure shown in Equation I: Formula I; Where 1≤n≤15.

2. The method for preparing the highly positively charged self-assembled ruthenium complex targeting and interfering with the electron transport chain as described in claim 1, characterized in that, Includes the following steps: 2-2-(pyridine)benzimidazole was reacted with compound 1 under alkaline conditions in organic solvent 1 to obtain ligand 1; 4,4'-bis(bromomethyl)-2,2'-bipyridine and trimethylamine were reacted in organic solvent 2 to obtain ligand 2; Under a protective atmosphere, cis -[RuCl2(DMSO)4] and ligand 1 undergo a coordination substitution reaction in a mixed solvent to obtain a ruthenium precursor complex containing ligand 1; The ruthenium precursor complex containing ligand 1 and ligand 2 undergo a ligand exchange reaction to obtain the highly positively charged self-assembled ruthenium complex. The structural formula of compound 1 is ; The structural formula of ligand 1 is ; The structural formula of ligand 2 is ; The structural formula of the ruthenium precursor complex containing ligand 1 is as follows: ; In compound 1, ligand 1, and the ruthenium precursor complex containing ligand 1, n is consistent with the n in the structural formula shown in Formula I.

3. The preparation method according to claim 2, characterized in that, The molar ratio of 2-2-(pyridine)benzimidazole to compound 1 is 1:1 to 1:3; the reagent providing the base in the alkaline conditions is potassium carbonate; the organic solvent 1 is N,N-dimethylformamide; the temperature of reaction 1 is 50 to 90°C, and the time is 6 to 10 hours.

4. The preparation method according to claim 2, characterized in that, The molar ratio of 4,4'-bis(bromomethyl)-2,2'-bipyridine to trimethylamine is 1:8 to 1:12; the organic solvent 2 is acetonitrile; the reaction 2 is carried out by stirring at room temperature for 0.5 to 2 hours and then drying by rotary evaporation.

5. The preparation method according to claim 2, characterized in that, The cis The molar ratio of -[RuCl2(DMSO)4] and ligand 1 is 1:1 to 1:3; the mixed solvent is a mixture of ethanol and dimethyl sulfoxide; the coordination substitution reaction is carried out at a temperature of 70 to 100 °C for 3 to 6 h.

6. The preparation method according to claim 2, characterized in that, The molar ratio of ligand 1 to ligand 2 is 1:1 to 1:3; the temperature of the ligand exchange reaction is 110 to 150°C, and the time is 8 to 13 hours.

7. The use of the highly positively charged self-assembled ruthenium complex that targets and interferes with the electron transport chain as described in claim 1 in the preparation of medicaments for the prevention and treatment of diseases caused by bacterial infections.

8. The application according to claim 7, characterized in that, The disease caused by bacterial infection is caused by methicillin-resistant Staphylococcus aureus infection.

9. A drug for the prevention and treatment of diseases caused by bacterial infections, characterized in that, Including the highly positively charged self-assembled ruthenium complex that targets and interferes with the electron transport chain as described in claim 1.

10. The medicament according to claim 9, characterized in that, The disease caused by bacterial infection is caused by methicillin-resistant Staphylococcus aureus infection.

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