Cu2O-BSO antibacterial nanoparticles and preparation method and application thereof

Abstract

The invention discloses a Cu2O-BSO antibacterial nano particle as well as a preparation method and an application of the Cu2O-BSO antibacterial nano particle. The preparation method comprises the following steps: (1) preparing Cu2O nanoparticles by adopting a Cu (NO3) 2 reduction method; (2) loading the sulphosulfoximine butyrate coated with dopa on the Cu2O nano particles obtained in the step (1), wherein the sulphosulfoximine butyrate coated with dopa is added into the Cu2O nano particles; and (3) adding polyethylene glycol into the product obtained in the step (2), and incubating to obtain the Cu2O-BSO antibacterial nanoparticles. The nano particles can effectively release Cu2O and BSO, the Cu2O and the BSO synergistically generate the effect of inducing bacterial copper death and the effect of inhibiting quorum sensing, the formation of a biological membrane and the expression of virulence factors can be influenced, and the synergistic antibacterial effect of non-antibiotics is not easily resisted by drug-resistant bacteria, so that drug-resistant pathogens can be effectively inhibited, and the drug resistance of the drug-resistant bacteria can be effectively inhibited. The compound can be used for treating diseases caused by drug-resistant bacterial infection.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical materials and specifically relates to a Cu 2 O-BSO antibacterial nanoparticles and preparation method and application thereof. Background Art

[0002] Methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa (PAO1) have antimicrobial resistance and high virulence, making the infections they cause more complicated and with higher mortality. MRSA produces many virulence factors, including α-hemolysin (Hla), phenol-soluble modulin (psm), exfoliative toxins, and leukocidins, which are essential for the spread and survival of bacteria in the host. PAO1 shows the ability to adapt to the harsh host environment by secreting a variety of virulence factors, including lipopolysaccharides, outer membrane proteins, flagella, adhesins, and a type 6 secretion system. In addition, biofilms are protective communities surrounded by a self-generated extracellular polymeric substance. The compact physical structure and the polysaccharides and nucleic acids contained in the extracellular polymeric substance (EPS) can serve as a defense barrier to inhibit the infiltration of antibiotics, leading to antibiotic resistance of bacteria in the biofilm matrix. Therefore, it is urgent to find new targets and new methods for broad-spectrum antimicrobial agents.

[0003] These virulence factors and biofilm regulatory mechanisms are mediated by quorum sensing (QS). QS is when the density of bacteria reaches a certain level, by releasing key QS autoinducers to help bacteria adapt to the harsh host environment and thrive, which can lead to persistent infection in patients even with major improvements in drugs, medical environment and living conditions. Therefore, it is imperative to continuously discover new methods and therapeutic targets to inhibit key QS systems and other key virulence factors of drug-resistant bacteria and remove biofilms.

[0004] Cuprous oxide nanoparticles (Cu 2 O NPs) can effectively catalyze H 2 O 2 The nanoparticles have been shown to promote the aggregation of lipoylated proteins and the degradation of Fe-S proteins by increasing the copper concentration, ultimately leading to copper death. In addition, these nanoparticles also exhibit anti-biofilm properties by generating reactive oxygen species (ROS) and electrostatically interacting with bacterial membranes. The presence of glutathione (GSH) in bacteria can serve as a protective mechanism against oxidative stress, thereby inhibiting the growth of Cu. 2 O NPs-induced copper death. Buthionine sulfoxide (BSO) is an inhibitor of γ-glutamylcysteine ​​synthetase (γ-GCS), inhibiting the biosynthesis of GSH, and is expected to promote the occurrence of copper death by effectively depleting intracellular GSH levels. 2The combined action of O NPs and BSO has the potential to synergistically induce copper poisoning-like bacterial death. However, the possibility of copper-like death in Gram-negative bacteria (such as Pseudomonas aeruginosa, PAO1) and the relationship between copper-like death and QS inhibition remain to be further investigated. Previous studies on copper-like bacterial death have mainly focused on superficial areas such as skin and bones, while ignoring deep tissues such as lungs. In addition, heavy metal poisoning caused by copper ions has obvious toxic effects on the liver and kidneys, which limits its application in systemic administration.

[0005] Bacterial pneumonia is considered a major public health problem due to its extremely high mortality and morbidity rates. MRSA and PAO1 are the main pathogens of acute bacterial pneumonia infections and pose a major threat to human health. Pulmonary inhalation is a non-invasive method of administering drugs through the throat and bronchi. Inhalation therapy can well control pulmonary administration and minimize systemic side effects, which has great potential in the treatment of various lung diseases (including tuberculosis, asthma, chronic obstructive pulmonary disease, and lung infections). However, in the case of pulmonary administration, the drug must pass through the mucus layer that wraps the lung mucosa to achieve absorption. Moreover, the lung's own clearance ability also makes the drug ineffective in the lungs. Summary of the invention

[0006] In view of the above-mentioned deficiencies in the prior art, the present invention provides a Cu 2 O-BSO antibacterial nanoparticles and their preparation method and application. 2 O nanoparticles were coated with DOPA and modified with PEG to form a new type of antibacterial microparticle preparation for airway delivery (Cu 2 O-BSO nanoparticles) increase the adhesion and mucosal penetration ability of the lungs, reduce systemic toxic side effects by local administration, and have the effect of inhibiting quorum sensing, which can show excellent antibacterial and anti-inflammatory effects and can ultimately be used to treat acute bacterial pneumonia.

[0007] The present invention has developed a Cu 2 Biocompatible Non-Antibiotic CuO Nanoparticles and BSO 2 O-BSO antimicrobial nanoparticles to combat a broad spectrum of difficult-to-treat bacteria, including Gram-positive bacteria such as MRSA and Gram-negative bacteria such as PAO1. 2 O-BSO antibacterial nanoparticles have excellent stability and can slowly release copper ions, and the release rate is further increased in the weakly acidic environment of inflammation.

[0008] The present invention quickly and conveniently synthesizes a special Cu 2 CuO nanoparticles and BSO 2O-BSO antibacterial nanoparticles can effectively release Cu and BSO, which can play a bactericidal role by inducing copper-like death. The particles have excellent broad-spectrum antibacterial effects and have excellent bactericidal effects on MRSA and PAO1. They can inhibit the formation of biofilms by 80% in the early stage and destroy 65% ​​of the formed biofilms in the later stage. Due to the modification of DOPA and PEG, the particles have the ability to adhere to the lungs and penetrate the mucosa. Due to local administration, the obtained nanoparticles have almost no systemic toxicity. No abnormalities were found in the blood routine, blood biochemistry, heart, liver, spleen, lung and kidney of BALB / C mice treated with the nanoparticles, indicating that the antibacterial nanoparticles have little effect on the safety of the body.

[0009] The Cu 2 O-BSO antibacterial nanoparticles can release Cu and BSO simultaneously, exerting a synergistic effect of inducing bacterial copper-like death and killing bacteria. BSO is an inhibitor of γ-GCS, which can inhibit the biosynthesis of GSH and increase the intracellular H 2 O 2 level, mediating a high level of Fenton reaction, ultimately promoting lipid peroxidation, amplifying bacterial copper death, and further killing bacteria. 2 O-BSO antibacterial nanoparticles can inhibit the quorum sensing (QS) system, thereby destroying the biofilm and reducing the expression of virulence factors, thereby inhibiting the bactericidal ability of bacteria and greatly improving the bactericidal efficiency. 2 O-BSO antibacterial nanoparticles showed excellent antibacterial effects both in vitro and in vivo, and also provided an effective therapeutic target and strategy for the treatment of bacterial pneumonia.

[0010] The present invention first provides a Cu 2 The preparation method of O-BSO antibacterial nanoparticles comprises the following steps:

[0011] (1) Using Cu(NO 3 ) 2 Preparation of Cu by reduction method 2 O nanoparticles;

[0012] (2) The Cu obtained in step (1) 2 O nanoparticles were loaded with DOPA-coated buthionine sulfoxide;

[0013] (3) Add polyethylene glycol to the product obtained in step (2) and incubate to obtain the Cu 2 O-BSO antibacterial nanoparticles.

[0014] Preferably, in step (1), Cu(NO 3 ) 2 , acrylic acid and NaOH to obtain the Cu 2O nanoparticles.

[0015] More preferably, Cu(NO 3 ) 2 Cu(NO 3 ) 2 ·3H 2 O, add 50-150 mg Cu(NO 3 ) 2 ·3H 2 O, 80-160 mg acrylic acid, 300-500 mg NaOH.

[0016] Preferably, Cu 2 The mass ratio of CuO nanoparticles to buthionine sulfoxide is 1:1 / 7 to 3. 2 The mass ratio of O nanoparticles to buthionine sulfoxide amine is 1:1 / 3-3.

[0017] Preferably, the mass ratio of buthionine sulfoxide to DOPA is 1:2.5-7.5; the mass ratio of buthionine sulfoxide to polyethylene glycol is 1:2.5-7.5.

[0018] Preferably, in step (2), the Cu 2 O nanoparticles were dispersed in water and then incubated with DOPA and buthionine sulfoxide.

[0019] Preferably, in step (3), the Cu 2 O-BSO antibacterial nanoparticles.

[0020] The present invention also provides Cu prepared by the preparation method 2 O-BSO antibacterial nanoparticles.

[0021] The present invention also provides the Cu 2 Application of O-BSO antibacterial nanoparticles in the preparation of broad-spectrum antibacterial and / or anti-inflammatory drugs.

[0022] The present invention also provides a broad-spectrum antibacterial and / or anti-inflammatory drug, the active ingredient of which is the Cu 2 O-BSO antibacterial nanoparticles.

[0023] The beneficial effects of the present invention are: the present invention designs a Cu with lung adhesion and mucosal penetration 2 O-BSO antibacterial nanoparticles. The nanoparticles can effectively release Cu 2O and BSO; unexpectedly, the two synergistically produce the induction of bacterial copper-like death effect and the inhibition of quorum sensing, which can affect the formation of biofilms and the expression of virulence factors. This non-antibiotic synergistic antibacterial effect is not easily resisted by drug-resistant bacteria, so it can effectively inhibit drug-resistant pathogens and can be used to treat diseases caused by drug-resistant bacterial infections. It has a killing effect on both Gram-positive and Gram-negative bacteria and has a broad-spectrum antibacterial effect. Its MIC for MRSA and PAO1 are 250μg / mL and 500μg / mL, respectively. In addition, the microparticles can significantly improve the survival rate of mice with acute severe bacterial pneumonia, reduce the inflammatory response in the lung lesions, and ultimately cure acute bacterial pneumonia. The systemic toxicity to other organs and tissues during the treatment process can be almost ignored. Therefore, the Cu 2 O-BSO antibacterial nanoparticles have become very potential antibacterial materials for the treatment of refractory bacterial infections such as bacterial pneumonia. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Cu 2 Characterization diagram of O-BSO antibacterial nanoparticles. Figure a: Cu 2 Schematic diagram of the synthesis of O-BSO antibacterial nanoparticles; b: Cu 2 O-BSO、PDA-Cu 2 O-BSO and Cu 2 Potential statistics of O-BSO nanoparticles; Figure c: Cu at different magnifications 2 O nanoparticles and Cu 2 Transmission electron microscopy (TEM) images of O-BSO nanoparticles; d, g: Cu 2 Energy spectrum analysis of O-BSO nanoparticles. Figure e: Release curves of copper ions at pH 6.0 and pH 7.4; Figure f: Cu ions at different magnifications 2 O nanoparticles and Cu 2 Scanning electron microscope (SEM) image of O-BSO nanoparticles.

[0025] Figure 2 Cu 2 The results of the lung retention and mucosal penetration test of O-BSO antibacterial nanoparticles. Figure a and b: 50 μL IR783-Cu was injected into the airway. 2 Representative in vivo and in vitro fluorescence images of mice at 6, 24, 48, and 96 hours after O-BSO antibacterial nanoparticles; Figure c: Quantitative analysis of the copper ion content in the artificial mucosa detected by the transwell method.

[0026] Figure 3The figures are the results of in vitro antibacterial activity test on drug-resistant bacteria, among which Figure a: Statistical graph of MRSA survival rate after different treatments; Figure b: Plate pictures of MRSA after different treatments; Figure c: Representative TEM images of MRSA after different treatments; Figure d: Statistical graph of PAO1 survival rate after different treatments; Figure e: Plate pictures of PAO1 after different treatments; Figure f: Representative TEM images of PAO1 after different treatments.

[0027] Figure 4 The results of in vitro anti-MRSA biofilm activity test are shown in Figure a. The 3D structure of the immature MRSA biofilm after different treatments at 0 hours was observed by laser confocal microscopy; Figure b. The 3D structure of the mature MRSA biofilm after different treatments at 24 hours was observed by laser confocal microscopy; Figure c. The 3D structure of the immature PAO1 biofilm after different treatments at 0 hours was observed by laser confocal microscopy; Figure d. The 3D structure of the mature PAO1 biofilm after different treatments at 24 hours was observed by laser confocal microscopy.

[0028] Figure 5 The results of the detection of the copper-like death phenotype of bacteria. Figure a: Statistical diagram of the lipid peroxidation level of MRSA after the corresponding treatment detected by the MDA kit; Figure b: GSH / GSSG ratio of MRSA in each group; Figure c: Copper ion content in MRSA in each group; Figure d: EDTA to Cu 2 Inhibitory effect of O-BSO antibacterial nanoparticles on bacterial death; eh graph: respiratory chain complex I-IV activity of MRSA in each group; i graph: pyruvate content of PAO1 in each group; j graph: GSH / GSSG ratio of PAO1 in each group; k graph: statistical graph of lipid peroxidation level of PAO1 after corresponding treatment detected by MDA kit; lo graph: respiratory chain complex I-IV activity of PAO1 in each group.

[0029] Figure 6 Cu 2 Results of the detection of the activation effect of O-BSO antibacterial nanoparticles on macrophages. Figure a: Typical scatter plot of macrophage surface markers CD86 (M1 macrophage marker) and CD206 (M2 macrophage marker) detected by flow cytometry; Figure b: Typical image of macrophages engulfing bacteria (green fluorescence indicates MRSA, red fluorescence indicates macrophages, and blue fluorescence indicates cell nuclei); Figures c and d: Representative plate photos and counting statistics of macrophages engulfing MRSA; Figure e: ELISA results showing the levels of inflammatory factors (IL-1β, IL-6, TNF-α) secreted by MH-S in different groups.

[0030] Figure 7 Cu 2The results of the in vivo antibacterial and anti-inflammatory effects of O-BSO antibacterial nanoparticles. 2 Schematic diagram of the experiment of O-BSO antibacterial nanoparticles in the treatment of acute bacterial pneumonia; b, c: blank TSB, Cu 2 O, BSO, Cu 2 Agar plate photos and MRSA quantitative analysis after 36 hours of treatment with O-BSO antibacterial nanoparticles; Figure d: Representative photos of the lungs of mice in each treatment group after infection; Figure eh: H&E staining of lung tissues in each group after infection (e), H&E scores of lung tissues (f), MPO staining (g) and NF-κB staining (h); Figure ik: Quantitative analysis of IL-1β, IL-6, and TNF-α levels in lung homogenates of different treatment groups after infection.

[0031] Figure 8 Cu 2 O, BSO, Cu 2 The preliminary toxicity test results of O-BSO. Figure ah: blood routine examination of healthy BALB / c mice in different groups on day 14; Figure il: blood biochemical analysis of healthy BALB / c mice in different groups on day 14; Figure m: H&E staining results of major organs (heart, liver, spleen, lung, kidney) of BALB / c mice in each group on day 14. DETAILED DESCRIPTION

[0032] Experimental Materials:

[0033] Cu(NO 3 ) 2 ·3H 2 O, acrylic acid (AA), and buthionine sulfoxide (BSO) were purchased from Aladdin, China. DOPA and polyethylene glycol (PEG, molecular weight 1000) were from Sigma-Aldrich (USA). Methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa (PAO1) were from the American Type Culture Collection (ATCC43300 and ATCC15692), respectively. TSB broth, TSB broth agar, and MDA detection kit were purchased from Solebol (Beijing, China). Fetal bovine serum (FBS) was purchased from Gibco. DMEM medium was purchased from Gibco (USA), and MH-S cell-specific medium was purchased from Purosai (Wuhan, China). Double distilled water (ddH 2 O) from Milli-Q purification system. BALB / c mice were from Shanghai Biotechnology Co., Ltd. Live / Dead Bacterial Viability Kit and DCFH-DA Kit were purchased from Thermo Fisher Scientific (China).

[0034] Characterization Instruments:

[0035] The surface potential was measured by dynamic light scattering (DLS) using a Malvern Zetasizer. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were used to observe the structure of these particles.

[0036] Example 1

[0037] (1)Cu 2 O-BSO antibacterial nanoparticles.

[0038] Cu 2 The synthesis process of O-BSO antibacterial nanoparticles is as follows Figure 1 As shown in a. Using Cu(NO 3 ) 2 Preparation of Cu by reduction method 2 O nanoparticles. 100 mg Cu(NO 3 ) 2 ·3H 2 O was dissolved in 90 mL ultrapure water, and then 10 mL AA (12 mg / mL) and 2 mL NaOH (200 mg / mL) were quickly added simultaneously. After reacting at room temperature for 30 min, the Cu was centrifuged at 8000 rpm to obtain 2 O nanoparticles were dispersed in 20 mL ultrapure water. Then 100 mg DOPA and 20 mg BSO were added and incubated overnight. Then 100 mg PEG was added and incubated at room temperature for 3 h. Finally, Cu was obtained by centrifugation. 2 O-BSO antibacterial nanoparticles. Among them, Cu 2 The O / BSO mass ratio is about 3:1.

[0039] (2) Characterization of properties.

[0040] We measured the surface potential of the particles. Figure 1 b shows that after PEG adsorption, Cu 2 The surface potential of O-BSO antibacterial nanoparticles is reduced, which is more conducive to penetrating the lung mucus barrier. Then SEM was used to observe the Cu 2 O nanoparticles and Cu 2 Morphology of O-BSO nanoparticles. Cu 2 O nanoparticles are regular cubes with a size of about 100 nm. After modification, the surface is covered with a polymer layer ( Figure 1 c). TEM further determined the Cu 2 The morphology and element distribution of O-BSO nanoparticles. The nanoparticles are basically cubic, which is consistent with SEM ( Figure 1 d in the figure). The results of energy spectrum analysis show that NPs contain elements such as Cu, O, S, C, and N, which are basically evenly distributed on the cube, further confirming that Cu 2The successful preparation of O and the successful loading of BSO ( Figure 1 In addition, we also measured the release curve of copper under different conditions. After modification, copper ions can be slowly released into the environment, and the release rate is further increased in the weakly acidic environment of inflammation ( Figure 1 g) in.

[0041] (3) Tissue adhesion and mucosal penetration ability.

[0042] Considering that DOPA has good tissue adhesion, we used IVIS imaging system to evaluate the 2 The residence time of O-BSO nanoparticles in the lung. 2 O-BSO nanoparticles were coupled and 50 μL was instilled into the mouse lungs via trachea. Fluorescence imaging was performed at 6h, 24h, 48h and 96h to analyze the fluorescence intensity of the lungs at each time point in vivo and in vitro. First, IR783-Cu was observed at 6h and 24h. 2 The fluorescence intensity of O-BSO nanoparticles in the lungs was strong, and at 48 h, there was still obvious fluorescence intensity in the lungs, indicating that Cu 2 O-BSO nanoparticles can stay in the lungs for more than 48 hours. At 96 hours, there is almost no fluorescence in the lungs, reflecting the Cu 2 O-BSO nanoparticles can be successfully degraded and metabolized in the body and will not cause long-term burden on the lungs ( Figure 2 The fluorescence intensity measured in vitro was highly similar to that measured in vivo. At 6h, 24h, and 48h, the fluorescence was mainly concentrated in the lungs. At 96h, no obvious fluorescence was observed in any organ, indicating that Cu 2 O-BSO nanoparticles were almost completely metabolized ( Figure 2 b). These results together indicate that Cu 2 O-BSO nanoparticles have good lung adhesion and good biodegradability.

[0043] Subsequently, we used an artificial mucus model to study the effect of PEG on Cu 2 Effect of O-BSO nanoparticles on mucosal permeability. Artificial airway mucus was prepared as follows: mucin and immunoglobulin sodium salt were dissolved in ultrapure water overnight under magnetic stirring. On the day of the experiment, mucin solution (final concentration of 43.75 mg / mL), calcium carbonate sodium salt solution (final concentration of 14 mg / mL), GDL (final concentration of 49.84 mg / mL) and AIg (final concentration of 21 mg / mL) were mixed in a reaction flask at room temperature under magnetic stirring for 1 min. Subsequently, equal amounts of PDA-Cu were added to the artificial mucus. 2 O-BSO (unmodified with PEG) or Cu 2O-BSO nanoparticles, the penetration of both in artificial mucus was recorded at 0, 0.5, 1, 1.5, 2 and 3.5 h. The Cu2O-BSO nanoparticle group had more copper ion penetration, indicating that its mucosal permeability was stronger ( Figure 2 c), and the penetration speed quantitative experiment shows that its penetration speed is about 2.1cm / h ( Figure 2 d) in the figure indicates mucosal penetration.

[0044] (4) Characterization of in vitro antibacterial properties.

[0045] In order to explore the antibacterial activity against MRSA, we obtained Cu 2 O-BSO nanoparticles were diluted to different concentrations (7.82, 15.63, 31.25, 62.5, 125, 250, 500 μg / mL), and then MRSA was incubated with different concentrations of Cu 2 After incubation with O-BSO nanoparticles for 24 hours, the OD 600 value was measured to calculate the minimum inhibitory concentration (MIC), and the MIC was 250 μL / mL ( Figure 3 Then, we further compared Cu 2 O, BSO and Cu 2 The anti-MRSA activity of O-BSO nanoparticles was recorded by agar plate counting method. We took 100 μL of bacterial solution, spread it evenly on the agar plate with a disposable spreader, inverted it in a 37°C incubator for 24 hours, and then counted the colonies. We can observe that Cu 2 The O-BSO nanoparticle group had the least number of colonies formed on the plate, showing excellent anti-MRSA effect ( Figure 3 b). We used TEM to observe the morphological changes of MRSA after treatment. TEM micrographs showed that MRSA treated with blank LB maintained a normal shape, with intact cell walls, smooth cell membranes, uniform and dense cytoplasm, and could form septa and split in the cell. However, Cu 2 O-BSO nanoparticle treatment can lead to cell wall rupture, cell membrane shrinkage and cytoplasm leakage ( Figure 3 c) in.

[0046] In order to explore the antibacterial activity against PAO1, we obtained Cu 2 O-BSO nanoparticles were diluted to different concentrations (62.5, 125, 250, 500 μg / mL), and then PAO1 was mixed with different concentrations of Cu 2 After incubation with O-BSO nanoparticles for 24 hours, the OD 600 value was measured to calculate the MIC, which was 500 μL / mL ( Figure 3d). Subsequently, we further compared the anti-PAO1 activity of each group, and we used the agar plate counting method to record the colony formation status and clone number of each group after the corresponding treatment. We can observe that Cu 2 The O-BSO nanoparticle group had the least number of colonies formed on the plate, showing excellent anti-PAO1 effect ( Figure 3 e). TEM results also show that Cu 2 O-BSO nanoparticles can destroy the integrity of the bacterial membrane and wall of PAO1, and the internal density of bacteria is greatly reduced ( Figure 3 f). In summary, these findings indicate that Cu 2 O-BSO nanoparticles have a promising future as a highly efficient broad-spectrum antibacterial material.

[0047] Next, we detected the anti-biofilm activity by SYTO 9 staining. In the biofilm disruption experiment, bacteria (1×10 7 CFU) were inoculated into a 24-well glass-bottomed culture plate and cultured at 37°C for 24 h. The supernatant was removed and replaced with blank TSB medium, medium containing BSO, and Cu 2 O and Cu 2 The culture medium was replaced with TSB medium containing O-BSO antibacterial particles, cultured at 37°C for 24 hours, washed with PBS three times, and then SYTO 9 fluorescent probe was added. The cells were stained at 37°C for 30 minutes and the biofilm 3D imaging was performed using a laser confocal microscope. In the biofilm inhibition experiment, bacteria were inoculated in a 24-well glass-bottomed culture plate containing blank TSB medium, and BSO, Cu 2 O and Cu 2 OBSO NPs. The subsequent steps were the same as the procedures for the biofilm disruption experiment. We compared the antibiofilm activities of the groups. As expected, the antibiofilm activity of MRSA biofilm was significantly inhibited by the Cu OBSO NPs at the early stage of MRSA biofilm formation (0 h). 2 After O-BSO nanoparticle treatment, it showed the weakest green signal and the thinnest thickness, which means that the ability to inhibit biofilm formation is the most prominent ( Figure 4 a). In the late stage of MRSA biofilm formation (24 hours), Cu 2 The biofilm was the most sparse and thin after O-BSO nanoparticle treatment, which means that the mature biofilm can be effectively destroyed ( Figure 4 b). In addition, Cu was given to PAO1 at the early and late stages of biofilm formation. 2 O-BSO nanoparticles treatment can show excellent anti-biofilm effect ( Figure 2 In conclusion, Cu 2 O-BSO nanoparticles all have significant anti-biofilm activity.

[0048] (5) In vitro bacterial copper-like death symptoms.

[0049] Next, we further explore Cu 2 Whether the antibacterial mechanism of O-BSO nanoparticles can be attributed to their induction of copper-like bacterial death. Malondialdehyde (MDA) experiments suggest that lipid peroxidation occurs in the MRSA membrane ( Figure 5 a) in the above formula, Figure 5 b in the figure shows that the ratio of oxidized glutathione (GSSG) to reduced glutathione (GSH) increased significantly, and the biological balance between GSSG and GSH was destroyed. The copper ion concentration experiment showed that copper ions accumulated significantly in MRSA ( Figure 5 c) The copper death inhibitor EDTA was added to Cu 2 O-BSO nanoparticles and incubated with MRSA. 2 When O-BSO nanoparticles are combined, their antibacterial effect can be inhibited, and the inhibition depends on the concentration of EDTA ( Figure 5 More importantly, the results of the respiratory chain detection kit indicated that Cu 2 O-BSO nanoparticles can inhibit MRSA respiratory chain complexes I-IV ( Figure 5 In addition, the results of pyruvate detection kit, GSSG / GSH detection kit, MDA detection kit and respiratory chain detection kit indicated that Cu 2 O-BSO nanoparticles can lead to pyruvate accumulation in PAO1 ( Figure 5 i), the ratio of GSSG and GSH increased significantly ( Figure 5 j), membrane lipids undergo peroxidation ( Figure 5 k in the figure), the activity of respiratory chains I-IV is inhibited ( Figure 5 The above results suggest that Cu 2 O-BSO nanoparticles can induce copper-like death in MRSA and PAO1.

[0050] (6) In vitro Cu 2 Activation effect of O-BSO nanoparticles on macrophages.

[0051] Mouse alveolar macrophages (MH-S) were cultured at 2×10 5 The cells were seeded at a density of 100 / well in a 6-well plate. After culturing at 37°C for 1 day, the cells were incubated with BSO, Cu 2 O and Cu 2O-BSO nanoparticles were replaced with fresh MH-S cell complete medium. After culturing at 37°C for 2 days, the cells were collected for flow cytometry analysis. Each group of cells was centrifuged, washed and resuspended in 100 μL PBS, and anti-mouse CD80 antibody conjugated with FITC and anti-mouse CD206 antibody conjugated with PE were added, incubated at 4°C in the dark for 30 minutes, and then the expression of CD86 and CD206 on the surface of MH-S cells was detected by flow cytometry. BSO, Cu 2 O and Cu 2 The percentages of M1 macrophages in the O-BSO nanoparticle group were 6.31%, 8.06%, and 8.64%, respectively ( Figure 6 These data show that Cu 2 O-BSO nanoparticles can promote macrophage polarization toward M1 phenotype. 2 The effect of O-BSO nanoparticle-mediated M1 polarization on macrophage function was evaluated by laser confocal microscopy (CLSM). MH-S cells were inoculated in confocal culture dishes and cultured at 37°C for 24 h. Afterwards, PBS, BSO, Cu 2 O and Cu 2 O-BSO nanoparticles were cultured for another 48 h. After adding GFP-labeled MRSA (MRSA: cells = 10-20:1), the culture was continued at 37 °C for 2 h. The culture dish was then washed three times with PBS, and fresh culture medium containing 200 μg / mL gentamicin was added to eliminate extracellular MRSA. After discarding the culture medium, the cells were washed, fixed, and permeabilized with Triton X-100, and then TRITC phalloidin and DAPI staining were applied, and all cell images were captured using CLSM. Compared with the other groups, Cu 2 O and Cu 2 The phagocytic ability of macrophages treated with O-BSO nanoparticles to MRSA was enhanced, and the Cu 2 O-BSO nanoparticles showed a more obvious effect, indicating that they have stronger phagocytic ability ( Figure 6 In addition, quantitative assessment of intracellular viable bacteria by plate spread counting further supported this conclusion ( Figure 6 c, d). We then evaluated the release of inflammatory cytokines by macrophages after the corresponding treatments by detecting the cell supernatant using an ELISA assay. 2 O and Cu 2 O-BSO nanoparticles can significantly increase the levels of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α, indicating that macrophages can exert stronger bactericidal activity through pro-inflammatory effects ( Figure 6 (eg).

[0052] (7)Cu 2 In vivo antibacterial and anti-inflammatory effects of O-BSO nanoparticles.

[0053] Encouraged by in vitro experiments, we further verified that Cu 2 Antibacterial and anti-inflammatory effects of O-BSO nanoparticles in vivo. Figure 7 a in Figure 1 is a schematic diagram of animal modeling. First, we established an acute pneumonia model by intratracheally injecting 50 μL of MRSA. Then, we injected PBS, BSO, Cu 2 O and Cu 2 O-BSO nanoparticles, and lung tissue was collected 24 hours after administration for subsequent experiments. Lung homogenate was plated and counted to assess the bacterial load in the lung. The results showed that Cu 2 The O-BSO nanoparticles treated group had the lowest bacterial colony count and the best antibacterial ability in vivo ( Figure 7 b, c).

[0054] After the antibacterial experiment, we detected Cu 2 The anti-inflammatory effect of O-BSO nanoparticles was further evaluated for its beneficial effects in the treatment of acute MRSA pneumonia. Similar to the PBS-treated group, mice in the BSO group showed obvious pulmonary hemorrhage and edema; in contrast, 2 O and Cu 2 The bleeding and edema in the O-BSO nanoparticle group were milder, and the Cu 2 The O-BSO nanoparticle group showed the mildest symptoms ( Figure 7 d). Subsequent hematoxylin and eosin (H&E) staining showed that the lung tissue structure of all groups was changed, including congestion of alveolar spaces, interstitial edema, and inflammatory cell infiltration. 2 The alveolar tissue structure of the O-BSO nanoparticle treatment group remained relatively clear, with only a small amount of edema and focal inflammatory cell infiltration in the alveolar cavity, and the degree of lung injury was significantly milder than that of the other groups ( Figure 7 e). Lung injury scores showed that the PBS and BSO groups had the most severe injury, followed by the Cu 2 O and Cu 2 O-BSO nanoparticles, and Cu 2 The O-BSO nanoparticle group had the least damage ( Figure 7 f) in.

[0055] Myeloperoxidase (MPO) is a heme peroxidase, and changes in its level and activity can serve as an indicator of neutrophil function and activation status and provide insights into the extent of neutrophil infiltration in lung tissue. Immunohistochemistry (IHC) analysis showed that Cu2 The O-BSO nanoparticle-treated group had the lowest MPO expression, indicating minimal neutrophil infiltration ( Figure 7 g). NF-κB is a protein complex that plays a key role in regulating the immune response to infection, and fluctuations in its level and activity may reflect the severity of lung tissue inflammation. 2 The O-BSO nanoparticles treatment group significantly reduced NF-κB expression and alleviated inflammation ( Figure 7 h). In addition, ELISA was used to assess the levels of proinflammatory cytokines, such as IL-1β, IL-6, and TNF-α, in lung tissue. 2 The O-BSO nanoparticle group had the lowest production of pro-inflammatory cytokines, indicating the lowest level of inflammation ( Figure 7 These results show that Cu 2 O-BSO nanoparticles can exert effective antibacterial and anti-inflammatory effects, protecting mice from lung damage caused by excessive inflammatory response, and ultimately improving the survival rate and prognosis of mice.

[0056] (8)Cu 2 Preliminary toxicity studies of O-BSO nanoparticles in vitro and in vivo.

[0057] In order to further promote the clinical application of antibacterial materials, a comprehensive evaluation of their toxicity is needed. 2 O and Cu 2 The potential long-term side effects of O-BSO nanoparticles. 50 μL BSO, Cu 2 O and Cu 2 On the 14th day after O-BSO, the retrobulbar venous blood of mice was collected to test blood routine and blood biochemistry. The results showed that blood biochemical indicators (such as glutamate-pyruvate aminotransferase (ALT), glutamate-oxalate aminotransferase (AST)) and blood routine indicators (such as white blood cells (WBC), red blood cells (RBC)) were within the normal range ( Figure 8 The tissue structure of major organs (including heart, liver, spleen, lungs and kidneys) remained normal, with no obvious inflammatory invasion, indicating good biological safety in vivo ( Figure 8 All these results provide strong evidence for biosafety, in other words, the antimicrobial materials we designed had no obvious negative effects on the hematopoietic system, liver and kidney functions, and major organs of mice.

[0058] Example 2-Example 10

[0059] Referring to the synthesis and detection methods of Example 1, various Cu 2O-BSO nanoparticles, the content is shown in Table 1, the concentrations in the table are the concentrations of the raw materials used in preparing this product, and the platelet membrane / erythrocyte membrane ratio is the total protein mass ratio. In Examples 2 to 10, only the corresponding parameters in each step were adjusted, and the adjusted parameters are shown in Table 1, and the other parameters were not adjusted.

[0060] Among them, lung adhesion: the residence time of the material in the lungs is detected by small animal living imaging. If it is greater than 24 hours, it is good, and if it is less than 24 hours, it is poor.

[0061] Mucosal penetration: Artificial mucus is prepared and the penetration rate of the material in the mucus is recorded for evaluation. A penetration rate greater than 2 cm / h is good, a penetration rate of 1-2 cm / h is average, and a penetration rate less than 1 cm / h is poor.

[0062] Material stability: Let the synthesized material stand for 24 hours. If there is no precipitation, it is good; if there is precipitation, it is bad.

[0063] Table 1

[0064]

[0065] The results show that under different reaction conditions, each Cu 2 O-BSO nanoparticles have good bactericidal effects. At the same time, the lung adhesion and mucosal penetration of antibacterial particles can be adjusted by adjusting the ratio of DOPA and PEG according to actual needs. 2 The ratio of O and BSO can adjust the material stability of the antimicrobial particles.

[0066] Test Example 1

[0067] Table 2

[0068] <![CDATA[Cu 2 O-BSO / μg / mL]]> Bactericidal rate (MRSA) Sterilization rate (PAO1) Cytotoxicity 125 45％ 27％ 2.8％ 250 95％ 53％ 3.5％ 500 98％ 90％ 8.9％ 1000 99％ 97％ 16.4％

[0069] Using the Cu in Example 1 2 After the O-BSO nanoparticles were diluted at different times, the bactericidal rate and cytotoxicity were tested. The results are shown in Table 2, indicating that as the concentration increases, the bactericidal rate also increases, but at the same time the cytotoxicity also increases. Therefore, it is necessary to select a suitable Cu 2 The concentration of O-BSO nanoparticles was adjusted to balance the relationship between bactericidal rate and cytotoxicity.

Claims

1. A method for preparing Cu2O-BSO antibacterial nanoparticles, characterized in that: The following steps are involved: (1) Cu2O nanoparticles were prepared by Cu(NO3)2 reduction method; (2) loading DOPA-encapsulated buthionine sulfoxide onto the Cu2O nanoparticles obtained in step (1); (3) Adding polyethylene glycol to the product obtained in step (2) and incubating to obtain the Cu2O-BSO antibacterial nanoparticles.

2. The method for preparing Cu2O-BSO antibacterial nanoparticles according to claim 1, characterized in that: In step (1), Cu(NO3)2, acrylic acid and NaOH are reacted to obtain the Cu2O nanoparticles.

3. The method for preparing Cu2O-BSO antibacterial nanoparticles according to claim 2, characterized in that: Cu(NO3)2 is Cu(NO3)2·3H2O. To every 90 mL of ultrapure water, add 50-150 mg of Cu(NO3)2·3H2O, 80-160 mg of acrylic acid, and 300-500 mg of NaOH.

4. The method for preparing Cu2O-BSO antibacterial nanoparticles according to claim 1, characterized in that: The mass ratio of Cu2O nanoparticles to buthionine sulfoxide is 1:1 / 7-3.

5. The method for preparing Cu2O-BSO antibacterial nanoparticles according to claim 1, characterized in that: The mass ratio of buthionine sulfoxide amine to dopa is 1:2.5-7.5; the mass ratio of buthionine sulfoxide amine to polyethylene glycol is 1:2.5-7.

5.

6. The method for preparing Cu2O-BSO antibacterial nanoparticles according to claim 1, characterized in that: In step (2), the Cu2O nanoparticles are dispersed in water, and then DOPA and buthionine sulfoxide are added for incubation.

7. The method for preparing the Cu2O-BSO antibacterial nanoparticles according to claim 1, characterized in that: In step (3), the Cu2O-BSO antibacterial nanoparticles are obtained by centrifugation after incubation.

8. Cu2O-BSO antibacterial nanoparticles prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the Cu2O-BSO antibacterial nanoparticles according to claim 8 in the preparation of broad-spectrum antibacterial and / or anti-inflammatory drugs.

10. A broad-spectrum antibacterial and / or anti-inflammatory drug, characterized in that: The active ingredient is the Cu2O-BSO antibacterial nanoparticles described in claim 8.

Images