A kind of Cu2O-BSO antibacterial nanoparticles, their preparation method and application

Abstract

The present invention discloses a Cu2O-BSO antibacterial nanoparticle, a preparation method thereof and an application. The preparation method comprises the following steps: (1) preparing Cu2O nanoparticles by a Cu(NO3)2 reduction method; (2) loading buthionine sulfoximine wrapped with dopamine on the Cu2O nanoparticles obtained in step (1); (3) incubating with polyethylene glycol in the product obtained in step (2) to obtain the Cu2O-BSO antibacterial nanoparticle. The nanoparticles of the present invention can effectively release Cu2O and BSO, and the two synergistically produce an effect of inducing cuproptosis in bacteria and inhibiting 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.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical materials, and particularly relates to a Cu2O-BSO antibacterial nanoparticle, a preparation method thereof and an 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 complex and with a higher mortality rate. MRSA produces many virulence factors, including α-hemolysin (Hla), phenol-soluble modulins (psm), exfoliative toxin and leukocidin, and these factors are crucial 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 lipopolysaccharide, outer membrane proteins, flagella, adhesins and type VI secretion system. In addition, a biofilm is a protective community surrounded by a self-generated extracellular polymer. The compact physical structure and the polysaccharides and nucleic acids contained in the extracellular polymer (EPS) can serve as a defense barrier to inhibit the infiltration of antibiotics, resulting in antibiotic resistance of bacteria in the biofilm matrix. Therefore, there is an urgent need to find new targets and new methods for broad-spectrum antibacterial.

[0003] These virulence factors and biofilm regulatory mechanisms are mediated by quorum sensing (QS). QS is that 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 will lead to persistent infections in patients even with significant improvements in drugs, medical environments and living conditions. Therefore, it is imperative to continuously discover new methods and therapeutic targets to inhibit the key QS system and other key virulence factors of drug-resistant bacteria and remove biofilms.

[0004] Cuprous oxide nanoparticles (Cu2O NPs) can effectively catalyze H2O2 at the inflammatory site and generate hydrogen radicals with strong antibacterial properties through the Fenton-like reaction, damaging the bacterial cell membrane. At the same time, these nanoparticles have been shown to promote the aggregation of lipoylated proteins and the degradation of Fe-S proteins by increasing copper concentration, ultimately leading to cuproptosis. In addition, these nanoparticles also exhibit anti-biofilm properties by generating reactive oxygen species (ROS) and electrostatic interactions with the bacterial membrane. The presence of glutathione (GSH) in bacteria can serve as a protective mechanism against oxidative stress, thus inhibiting cuproptosis induced by Cu2O NPs. Buthionine sulfoximine (BSO) is an inhibitor of γ-glutamylcysteine synthetase (γ-GCS), which inhibits the biosynthesis of GSH and is expected to promote the occurrence of cuproptosis by effectively depleting intracellular GSH levels. The combined action of Cu2O NPs and BSO has the potential to synergistically induce cuproptosis-like bacterial death. However, the possibility of cuproptosis-like occurrence in Gram-negative bacteria (such as Pseudomonas aeruginosa, PAO1) and the relationship between cuproptosis-like and QS inhibition still need to be further studied. Previous studies on cuproptosis-like bacterial death mainly focused on superficial areas such as the skin and bones, while ignoring deep tissues such as the 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 drug delivery.

[0005] Bacterial pneumonia is regarded as a major public health problem due to its extremely high mortality and morbidity. MRSA and PAO1 are the main pathogens of acute bacterial pneumonia infection, posing a major threat to human health. Pulmonary inhalation is a non-invasive method of drug delivery through the throat and bronchi. Inhalation therapy can well control pulmonary drug delivery and minimize systemic side effects, which has great potential in the treatment of various pulmonary diseases (including tuberculosis, asthma, chronic obstructive pulmonary disease, and pulmonary infections). However, in the case of pulmonary drug delivery, the drug must penetrate the mucus layer covering the lung mucosa to be absorbed. Moreover, the self-clearance ability of the lungs also makes the drug ineffective in the lungs. Summary of the Invention

[0006] The present invention addresses the above deficiencies in the prior art and provides a Cu2O-BSO antibacterial nanoparticle, its preparation method, and application. BSO wrapped with DOPA on Cu2O nanoparticles and modified with PEG to form a novel antibacterial microparticle preparation for airway administration (Cu2O-BSO nanoparticles), which increases the adhesion and mucosal penetration ability in the lungs, reduces systemic toxic side effects by local drug delivery, and has the effect of inhibiting quorum sensing, and can exhibit excellent antibacterial and anti-inflammatory effects, and is ultimately used for the treatment of acute bacterial pneumonia.

[0007] The present invention develops biocompatible non - antibiotic Cu2O - BSO antibacterial nanoparticles containing Cu2O nanoparticles and BSO to combat a broad spectrum of refractory bacteria, including Gram - positive bacteria such as MRSA and Gram - negative bacteria such as PAO1. The Cu2O - BSO antibacterial nanoparticles have excellent stability, 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 Cu2O - BSO antibacterial nanoparticle that simultaneously contains Cu2O nanoparticles and BSO, which can effectively release Cu and BSO, and play a bactericidal role by inducing cuproptosis. This particle has excellent broad - spectrum antibacterial effects, has excellent bactericidal effects on both MRSA and PAO1, can inhibit the formation of ~80% of the biofilm in the early stage, and can destroy ~65% of the already formed biofilm in the later stage. Due to the modification of DOPA and PEG, the particle has the ability of lung adhesion and mucosal penetration. And due to local administration, the obtained nanoparticles have almost no systemic toxicity. The blood routine, blood biochemistry, and heart, liver, spleen, lungs, and kidneys of BALB / C mice treated with these nanoparticles were not found to be abnormal, indicating that the antibacterial nanoparticles have basically no impact on the safety of the body.

[0009] The Cu2O - BSO antibacterial nanoparticles described in the present invention can simultaneously release Cu and BSO, playing a synergistic role in inducing cuproptosis and bactericidal effects in bacteria. BSO is an inhibitor of γ - GCS, which can inhibit the biosynthesis of GSH, increase the intracellular H2O2 level, mediate a high - level Fenton reaction, ultimately promote lipid peroxidation, amplify cuproptosis in bacteria, and further kill bacteria. In addition, Cu2O - BSO antibacterial nanoparticles can disrupt biofilms by inhibiting the quorum - sensing (QS) system, thereby reducing the expression of virulence factors and further inhibiting the bactericidal ability of bacteria, greatly improving the bactericidal efficiency. Cu2O - BSO antibacterial nanoparticles show excellent antibacterial effects both in vitro and in vivo, and also provide effective therapeutic targets and strategies for the treatment of bacterial pneumonia.

[0010] The present invention first provides a preparation method of Cu2O - BSO antibacterial nanoparticles, including the following steps:

[0011] (1) Prepare Cu2O nanoparticles by the Cu(NO3)2 reduction method;

[0012] (2) Load DOPA - encapsulated buthionine sulfoximine on the Cu2O nanoparticles obtained in step (1);

[0013] (3) Add polyethylene glycol to the product obtained in step (2) and incubate to obtain the Cu2O - BSO antibacterial nanoparticles.

[0014] Preferably, in step (1), Cu2O nanoparticles are prepared by reacting Cu(NO3)2, acrylic acid and NaOH.

[0015] More preferably, Cu(NO3)2 is Cu(NO3)2·3H2O, and 50 - 150 mg of Cu(NO3)2·3H2O, 80 - 160 mg of acrylic acid, and 300 - 500 mg of NaOH are added to every 90 mL of ultrapure water.

[0016] Preferably, the mass ratio of Cu2O nanoparticles to buthionine sulfoximine is 1∶1 / 7 - 3. More preferably, the mass ratio of Cu2O nanoparticles to buthionine sulfoximine is 1∶1 / 3 - 3.

[0017] Preferably, the mass ratio of buthionine sulfoximine to dopa is 1∶2.5 - 7.5; the mass ratio of buthionine sulfoximine to polyethylene glycol is 1∶2.5 - 7.5.

[0018] Preferably, in step (2), the Cu2O nanoparticles are dispersed in water, and then dopa and buthionine sulfoximine are added for incubation.

[0019] Preferably, in step (3), the Cu2O - BSO antibacterial nanoparticles are obtained by centrifugation after incubation.

[0020] The present invention also provides Cu2O - BSO antibacterial nanoparticles prepared by the above preparation method.

[0021] The present invention also provides the application of the Cu2O - 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 Cu2O - BSO antibacterial nanoparticles.

[0023] The beneficial effects of the present invention are as follows: The present invention designs a Cu2O-BSO antibacterial nanoparticle with lung adhesion and mucosal penetration ability. This nanoparticle can effectively release Cu2O and BSO; unexpectedly, the two work synergistically to produce an induced cuproptosis effect in bacteria and inhibit 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. Moreover, it has a killing effect on both Gram-positive bacteria and Gram-negative bacteria, showing a broad-spectrum antibacterial effect. Its MIC values for MRSA and PAO1 are 250 μg / mL and 500 μg / mL respectively. In addition, this microparticle can significantly improve the survival rate of mice with acute severe bacterial pneumonia, reduce the inflammatory response at the lung lesion site, and ultimately cure acute bacterial pneumonia. The systemic toxicity to other organ tissues during the treatment process can be almost ignored. Therefore, the Cu2O-BSO antibacterial nanoparticle of the present invention becomes a very promising antibacterial material for treating refractory bacterial infections such as bacterial pneumonia. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a characterization diagram of the Cu2O-BSO antibacterial nanoparticle. Among them, Figure a: Schematic diagram of the synthesis of the Cu2O-BSO antibacterial nanoparticle; Figure b: Potential statistical diagram of the Cu2O-BSO, PDA-Cu2O-BSO, and Cu2O-BSO nanoparticles; Figure c: Transmission electron microscope (TEM) images of the Cu2O nanoparticles and Cu2O-BSO nanoparticles at different magnifications; Figures d and g: Energy spectrum analysis of the Cu2O-BSO nanoparticles. Figure e: Release curves of copper ions at pH 6.0 and pH 7.4; Figure f: Scanning electron microscope (SEM) images of the Cu2O nanoparticles and Cu2O-BSO nanoparticles at different magnifications.

[0025] Figure 2 It is a detection result diagram of the lung retention ability and mucosal penetration ability of the Cu2O-BSO antibacterial nanoparticle. Among them, Figures a and b: Representative fluorescence images in mice and in vitro fluorescence images at 6, 24, 48, and 96 h after intratracheal injection of 50 μL of IR783-Cu2O-BSO antibacterial nanoparticle; Figure c: Quantitative analysis of the copper ion content penetrating the artificial mucosa detected by the transwell method; Figure d: Detection result of the penetration speed quantitative experiment.

[0026] Figure 3It is a graph showing the in vitro antibacterial activity test results against drug-resistant bacteria. Among them, Figure a: Statistical graph of the survival rate of MRSA after different treatments; Figure b: Plating pictures of MRSA after different treatments; Figure c: Representative TEM images of MRSA after different treatments; Figure d: Statistical graph of the survival rate of PAO1 after different treatments; Figure e: Plating pictures of PAO1 after different treatments; Figure f: Representative TEM images of PAO1 after different treatments.

[0027] Figure 4 It is a graph showing the in vitro anti-MRSA biofilm activity test results. Among them, Figure a: 3D structure of immature MRSA biofilm after different treatments observed by laser confocal microscopy at 0 hour; Figure b: 3D structure of mature MRSA biofilm after different treatments observed by laser confocal microscopy at 24 hours; Figure c: 3D structure of immature PAO1 biofilm after different treatments observed by laser confocal microscopy at 0 hour; Figure d: 3D structure of mature PAO1 biofilm after different treatments observed by laser confocal microscopy at 24 hours.

[0028] Figure 5 It is a graph showing the detection results of the cuproptosis phenotype in bacteria. Among them, Figure a: Statistical graph of the lipid peroxidation level of MRSA detected by MDA kit after corresponding treatments; Figure b: GSH / GSSG ratio of each group of MRSA; Figure c: Copper ion content inside each group of MRSA; Figure d: Inhibitory effect of EDTA on the bacterial death induced by Cu2O-BSO antibacterial nanoparticles; Figures e-h: Activities of respiratory chain complexes I-IV of each group of MRSA; Figure i: Pyruvate content of each group of PAO1; Figure j: GSH / GSSG ratio of each group of PAO1; Figure k: Statistical graph of the lipid peroxidation level of PAO1 detected by MDA kit after corresponding treatments; Figures l-o: Activities of respiratory chain complexes I-IV of each group of PAO1.

[0029] Figure 6 It is a graph showing the detection results of the activation effect of Cu2O-BSO antibacterial nanoparticles on macrophages. Among them, 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 macrophage phagocytosis of bacteria (green fluorescence represents MRSA, red fluorescence represents macrophages, and blue fluorescence represents cell nuclei); Figures c and d: Representative plating photos and counting statistical graphs of macrophage phagocytosis of MRSA; Figures e-g: ELISA results showing the levels of inflammatory factors (IL-1β, IL-6, TNF-α) secreted by MH-S in different groups.

[0030] Figure 7Detection results of in vivo antibacterial and anti-inflammatory effects of Cu2O-BSO antibacterial nanoparticles. Among them, Figure a: Schematic diagram of the experiment of treating acute bacterial pneumonia with Cu2O-BSO antibacterial nanoparticles; Figures b and c: Photos of agar plates and quantitative analysis of MRSA after treatment with blank TSB, Cu2O, BSO, and Cu2O-BSO antibacterial nanoparticles for 36 h; Figure d: Representative photos of the lungs of mice in each treatment group after infection; Figures e-h: H&E staining of lung tissues in each group after infection (e), H&E score of lung tissues (f), MPO staining (g), and NF-κB staining (h); Figures i-k: Quantitative analysis of the levels of IL-1β, IL-6, and TNF-α in lung homogenates of different treatment groups after infection.

[0031] Figure 8 Detection results of the preliminary toxicity of Cu2O, BSO, and Cu2O-BSO. Among them, Figures a-h: Routine blood tests of healthy BALB / c mice in different groups on the 14th day; Figures i-l: Blood biochemical analysis of healthy BALB / c mice in different groups on the 14th day; Figure m: Results of H&E staining of the main organs (heart, liver, spleen, lung, kidney) of BALB / c mice in each group on the 14th day. Specific implementation manners

[0032] Experimental materials:

[0033] Cu(NO3)2·3H2O, acrylic acid (AA), and buthionine sulfoximine (BSO) were purchased from Aladdin (China). Dopamine (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 kits were purchased from Solarbio (Beijing, China). Fetal bovine serum (FBS) was purchased from Gibco. DMEM medium was purchased from Gibco (USA), and the special medium for MH-S cells was purchased from Procell (Wuhan, China). Double-distilled water (ddH2O) was from the Milli-Q purification system. BALB / c mice were from Shanghai Biotechnology Co., Ltd. The 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) Cu2O-BSO antibacterial nanoparticles.

[0038] The synthesis process of Cu2O-BSO antibacterial nanoparticles is as shown in a of Figure 1 . Cu2O nanoparticles were prepared by the Cu(NO3)2 reduction method. 100 mg of Cu(NO3)2·3H2O was dissolved in 90 mL of ultrapure water, and then 10 mL of AA (12 mg / mL) and 2 mL of NaOH (200 mg / mL) were rapidly added simultaneously. After reacting at room temperature for 30 min, the Cu2O nanoparticles were obtained by centrifugation at 8000 rpm and then redispersed in 20 mL of ultrapure water. Then, 100 mg of DOPA and 20 mg of BSO were added and incubated overnight. Then, 100 mg of PEG was added and incubated at room temperature for 3 h. Finally, the Cu2O-BSO antibacterial nanoparticles were obtained by centrifugation. Among them, the mass ratio of Cu2O / BSO is about 3:1.

[0039] (2) Characterization of properties.

[0040] We measured the surface potential of the particles. Figure 1 As shown in b of Figure 1 , after adsorbing PEG, the surface potential of the Cu2O-BSO antibacterial nanoparticles decreased, which was more conducive to penetrating the pulmonary mucus barrier. Then, the morphologies of the Cu2O nanoparticles and the Cu2O-BSO nanoparticles were observed by SEM. The Cu2O nanoparticles were regular cubes with a size of about 100 nm, and a polymer layer covered the surface after modification ( Figure 1 in c of Figure 1 ). TEM further determined the morphology and element distribution of the Cu2O-BSO nanoparticles. The nanoparticles were basically cube-shaped, which was consistent with SEM ( Figure 1 in d of

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

[0042] Considering the good tissue adhesion of DOPA, we used the IVIS imaging system to evaluate the residence time of Cu2O-BSO nanoparticles in the lungs. The IR783 dye was conjugated with Cu2O-BSO nanoparticles and 50 μL was instilled into the lungs of mice via trachea. Fluorescence imaging was performed at 6 h, 24 h, 48 h, and 96 h, respectively, and the fluorescence intensity in the lungs at each time point in vivo and ex vivo was analyzed. First, strong fluorescence intensity of IR783-Cu2O-BSO nanoparticles in the lungs was observed at 6 h and 24 h. By 48 h, there was still obvious fluorescence intensity in the lungs, indicating that Cu2O-BSO nanoparticles could stay in the lungs for more than 48 h. By 96 h, there was almost no fluorescence in the lungs, reflecting that Cu2O-BSO nanoparticles could be successfully degraded and metabolized in vivo without causing a long-term burden on the lungs( Figure 2 a) in. The fluorescence intensity measured ex vivo was highly similar to that measured in vivo, and the fluorescence was mainly concentrated in the lungs at 6 h, 24 h, and 48 h. At 96 h, no obvious fluorescence was seen in each organ, suggesting that Cu2O-BSO nanoparticles were almost completely metabolized( Figure 2 b) in. These results together indicate that Cu2O-BSO nanoparticles have good lung adhesion and good biodegradability.

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

[0044] (4) In vitro antibacterial property characterization.

[0045] To explore the antibacterial activity against MRSA, we diluted the obtained Cu2O-BSO nanoparticles into different concentrations (7.82, 15.63, 31.25, 62.5, 125, 250, 500 μg / mL). Subsequently, MRSA was co-incubated with different concentrations of Cu2O-BSO nanoparticles for 24 hours, and then the OD600 value was measured to calculate the minimum inhibitory concentration (MIC), and the MIC was found to be 250 μL / mL ( Figure 3 as shown in a) of Figure 3 . Subsequently, we further compared the anti-MRSA activities of the Cu2O, BSO, and Cu2O-BSO nanoparticle groups. We used the agar plate counting method to record the colony formation status and the number of clones in each group after the corresponding treatment. We took 100 μL of the bacterial solution, evenly spread it on the agar plate with a disposable spreading rod, and then inverted it and cultured it in an incubator at 37 °C for 24 h before performing colony counting. We could observe that the number of colonies formed on the plate of the Cu2O-BSO nanoparticle group was the least, showing excellent anti-MRSA effects ( Figure 3 as shown in b) of

[0046] To explore the antibacterial activity against PAO1, we diluted the obtained Cu2O-BSO nanoparticles into different concentrations (62.5, 125, 250, 500 μg / mL). Subsequently, PAO1 was co-incubated with different concentrations of Cu2O-BSO nanoparticles for 24 hours, and then the OD600 value was measured to calculate the MIC, and the MIC was 500 μL / mL ( Figure 3 as shown in d) of Figure 3 . Subsequently, we further compared the anti-PAO1 activities of each group. We used the agar plate counting method to record the colony formation status and the number of clones in each group after the corresponding treatment. We could observe that the number of colonies formed on the plate of the Cu2O-BSO nanoparticle group was the least, showing excellent anti-PAO1 effects ( Figure 3 as shown in e) of

[0047] In conclusion, these findings all indicate that Cu2O-BSO nanoparticles have a promising future as an 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 24-well glass-bottom culture plates. After culturing at 37 °C for 24 h, the supernatant was removed and replaced with blank TSB medium, TSB medium containing BSO, Cu2O, and Cu2O-BSO antibacterial particles, respectively. After culturing at 37 °C for 24 h, it was washed 3 times with PBS, and then SYTO 9 fluorescent probe was added. After staining at 37 °C for 30 min, 3D imaging of biofilms was performed using a laser confocal microscope. In the biofilm inhibition experiment, bacteria were inoculated into 24-well glass-bottom culture plates containing blank TSB medium, and BSO, Cu2O, and Cu2O-BSO NPs were added simultaneously. The subsequent steps were the same as those of the biofilm disruption experiment. We compared the anti-biofilm activities of each group. As expected, after treatment with Cu2O-BSO nanoparticles at the early stage (0 h) of MRSA biofilm formation, the weakest green signal and the thinnest thickness were shown, indicating the most prominent ability to inhibit biofilm formation ( Figure 4 a) in Figure 4 . After treatment with Cu2O-BSO nanoparticles at the late stage (24 h) of MRSA biofilm formation, the biofilm was the sparest and thinnest, indicating that mature biofilms could be effectively disrupted ( Figure 2 b) in

[0048] . In addition, when Cu2O-BSO nanoparticles were used to treat the early and late stages of PAO1 biofilm formation respectively, excellent anti-biofilm effects could be seen (

[0049] c, d) in Figure 5 . In summary, Cu2O-BSO nanoparticles have significant anti-biofilm activities both at the early and late stages of MRSA and PAO1 biofilm formation. Figure 5 . Figure 5 b showed that the ratio of oxidized glutathione (GSSG) to reduced glutathione (GSH) increased significantly, and the biological balance between GSSG and GSH was disrupted. The copper ion concentration experiment showed that copper ions accumulated significantly in MRSA ( Figure 5 c) in Figure 5in e-h). In addition, the results of the pyruvate detection kit, GSSG / GSH detection kit, MDA detection kit and respiratory chain detection kit respectively indicate that Cu2O-BSO nanoparticles can cause pyruvate accumulation in PAO1 ( Figure 5 in i), the ratio of GSSG and GSH increases significantly ( Figure 5 in j), membrane lipid peroxidation occurs ( Figure 5 in k), and the activities of respiratory chain I-IV are inhibited ( Figure 5 in l-o). The above results all indicate that Cu2O-BSO nanoparticles can induce cuproptosis-like phenomena in MRSA and PAO1.

[0050] (6) Activation of macrophages by Cu2O-BSO nanoparticles in vitro.

[0051] Mouse alveolar macrophages (MH-S) were seeded in 6-well plates at a density of 2×10 5 cells / well. After culturing at 37 °C for 1 day, the original medium was replaced with fresh MH-S cell complete medium containing BSO, Cu2O and Cu2O-BSO nanoparticles. After culturing at 37 °C for 2 days, the cells were collected for flow cytometry analysis. Cells in each group were centrifuged, washed and resuspended in 100 μL PBS, and at the same time, anti-mouse CD80 antibody conjugated with FITC and anti-mouse CD206 antibody conjugated with PE were added, and incubated at 4 °C in the dark for 30 min, and then the expression of CD86 and CD206 on the surface of MH-S cells was detected by flow cytometry. The percentages of M1 macrophages in the BSO, Cu2O and Cu2O-BSO nanoparticle groups were 6.31%, 8.06% and 8.64% respectively ( Figure 6in a). These data indicate that Cu2O-BSO nanoparticles can promote macrophage polarization towards the M1 phenotype. To further investigate the effect of Cu2O-BSO nanoparticle-mediated M1 polarization on macrophage function, we used confocal laser scanning microscopy (CLSM) to evaluate macrophage phagocytosis. MH-S cells were seeded in confocal dishes and cultured at 37 °C for 24 h. Subsequently, PBS, BSO, Cu2O, and Cu2O-BSO nanoparticles were added and the cells were cultured for an additional 48 h. After adding GFP-labeled MRSA (MRSA: cells = 10 - 20:1), the cells were cultured at 37 °C for 2 h. Then, the dishes were washed three times with PBS, and fresh medium containing 200 μg / mL gentamicin was added to eliminate extracellular MRSA. After discarding the medium, the cells were washed, fixed, and permeabilized with Triton X-100, and then stained with TRITC-phalloidin and DAPI, and all cell images were captured using CLSM. Compared with other groups, macrophages treated with Cu2O and Cu2O-BSO nanoparticles showed enhanced phagocytic ability towards MRSA, and Cu2O-BSO nanoparticles showed a more obvious effect, indicating stronger phagocytic ability ( Figure 6 in b). In addition, quantitative evaluation of viable bacteria inside the cells by the plate spreading counting method further supported this conclusion ( Figure 6 in c, d). Subsequently, we evaluated the release of inflammatory cytokines by macrophages after corresponding treatments by detecting the cell supernatants using enzyme-linked immunosorbent assay (ELISA) experiments. Both Cu2O and Cu2O-BSO nanoparticles significantly increased 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 in e-g).

[0052] (7) In vivo antibacterial and anti-inflammatory effects of Cu2O-BSO nanoparticles.

[0053] Encouraged by the in vitro experiments, we further verified the antibacterial and anti-inflammatory effects of Cu2O-BSO nanoparticles in vivo. Figure 7 a in is a schematic diagram of animal model establishment. First, we established an acute pneumonia model by intratracheal injection of 50 μL MRSA. Subsequently, PBS, BSO, Cu2O, and Cu2O-BSO nanoparticles were injected intratracheally, and lung tissues were collected 24 h after administration for subsequent experiments. Lung homogenates were plated for counting to evaluate the bacterial load in the lungs. The results showed that the Cu2O-BSO nanoparticle treatment group had the fewest bacterial colonies and the best antibacterial ability in vivo ( Figure 7 in b, c).

[0054] After the antibacterial experiment, we detected the anti-inflammatory effect of Cu2O-BSO nanoparticles and further evaluated their beneficial effects in the treatment of acute MRSA pneumonia. Similar to the PBS-treated group, obvious pulmonary hemorrhage and edema were observed in the BSO group; in contrast, the degrees of hemorrhage and edema in the Cu2O and Cu2O-BSO nanoparticle groups were milder, and the Cu2O-BSO nanoparticle group showed the mildest symptoms ( Figure 7 d) in Figure 7 . Subsequently, hematoxylin and eosin (H&E) staining showed that the lung tissue structure changed in all groups, including alveolar interstitial congestion, interstitial edema, and inflammatory cell infiltration. Notably, the alveolar tissue structure in the Cu2O-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 less than that in other groups ( Figure 7 e) in

[0055] . Lung injury scores showed that the PBS and BSO groups had the most severe injuries, followed by the Cu2O and Cu2O-BSO nanoparticle groups, and the Cu2O-BSO nanoparticle group had the least injury ( Figure 7 f) in Figure 7 . Figure 7 Myeloperoxidase (MPO) is a heme peroxidase, and changes in its level and activity can be used as an indicator of neutrophil function and activation status and provide insights into the degree of neutrophil infiltration in lung tissue. Immunohistochemical (IHC) analysis showed that the MPO expression in the Cu2O-BSO nanoparticle treatment group was the lowest, indicating the least neutrophil infiltration (

[0056] g) in

[0057] Figure 7 . 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. Accordingly, IHC analysis showed that the NF-κB expression in the Cu2O-BSO nanoparticle treatment group was significantly decreased and the inflammation was alleviated ( Figure 7 h) in Figure 7 . In addition, ELISA was used to evaluate the levels of pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α in lung tissue. The results showed that the production of pro-inflammatory cytokines in the Cu2O-BSO nanoparticle group was the lowest, indicating the lowest level of inflammation ( Figure 7 i-k) in

[0056] . These results indicate that Cu2O-BSO nanoparticles can exert effective antibacterial and anti-inflammatory effects, protect mice from lung injury caused by excessive inflammatory responses, and ultimately improve the survival rate and prognosis of mice.

[0056] (8) Preliminary toxicity study of Cu2O-BSO nanoparticles in vitro and in vivo.

[0057] To further promote the clinical application of antibacterial materials, it is necessary to comprehensively evaluate their toxicity. The long-term potential side effects of BSO, Cu2O, and Cu2O-BSO nanoparticles were evaluated in healthy mice. Retroorbital venous blood of mice was collected on the 14th day after 50 μL of BSO, Cu2O, and Cu2O-BSO were instilled into the lungs of mice respectively to detect blood routine and blood biochemistry. The results showed that blood biochemical indexes (such as glutamate-pyruvate transaminase (ALT), glutamate-oxaloacetate transaminase (AST)) and blood routine indexes (such as white blood cells (WBC), red blood cells (RBC)) were within the normal range ( Figure 8 in a-l of). The organizational structures of the main organs (including the heart, liver, spleen, lungs, and kidneys) remained normal without obvious inflammatory invasion, indicating good biosafety in vivo ( Figure 8 in m of). All these results provide strong evidence for biosafety. In other words, the antibacterial materials we designed have no obvious negative effects on the hematopoietic system, liver and kidney functions, and main organs of mice.

[0058] Examples 2 - 10

[0059] Referring to the synthesis and detection methods of Example 1, various Cu2O-BSO nanoparticles were synthesized in Examples 2 - 10. The details are shown in Table 1. The concentrations in the table are the usage concentrations of raw materials when preparing this product, and the platelet membrane / red blood cell membrane ratio is the total protein mass ratio. In Examples 2 - 10, only the corresponding parameters in each step were adjusted, and the adjusted parameters are shown in Table 1, while the other parameters were not adjusted.

[0060] Among them, pulmonary adhesion: The residence time of the material in the lungs was detected by small animal in vivo imaging. More than 24 hours is good, and less than 24 hours is poor.

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

[0062] Material stability: The synthesized material was left standing for 24 hours. No precipitation is good, and precipitation is poor.

[0063] Table 1

[0064]

[0065] The results showed that each Cu2O-BSO nanoparticle under different reaction conditions had good bactericidal effects. At the same time, according to actual needs, the pulmonary adhesion and mucosal penetration of antibacterial particles could be adjusted by adjusting the ratio of DOPA and PEG, and the material stability of antibacterial particles could be adjusted by adjusting the ratio of Cu2O and BSO.

[0066] Detection Example 1

[0067] Table 2

[0068] <![CDATA[Cu2O-BSO / μg / mL]]> Bactericidal rate (MRSA) Bactericidal rate (PAO1) Cytotoxicity 125 45％ 27％ 2.8％ 250 95％ 53％ 3.5％ 500 98％ 90％ 8.9％ 1000 99％ 97％ 16.4％

[0069] After diluting the Cu2O-BSO nanoparticles in Example 1 by different multiples, the bactericidal rate and cytotoxicity were detected. 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 an appropriate concentration of Cu2O-BSO nanoparticles to balance the relationship between the bactericidal rate and cytotoxicity.

Claims

1. A method for preparing Cu2O-BSO antibacterial nanoparticles, characterized in that: The following steps are involved: (1) Preparation of Cu2O nanoparticles 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 the mixture to obtain the Cu2O-BSO antibacterial nanoparticles; The mass ratio of Cu2O nanoparticles to buthionine sulfoxide amine is 1:1 / 3~3; The mass ratio of buthionine sulfoxide to dopa is 1:5-7.5; the mass ratio of buthionine sulfoxide to polyethylene glycol is 1:2.5-7.

5.

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. Add 50~150 mg Cu(NO3)2·3H2O, 80~160 mg acrylic acid, and 300~500 mg NaOH to every 90 mL ultrapure water.

4. 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.

5. The method for preparing 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.

6. Cu2O-BSO antibacterial nanoparticles prepared by the preparation method according to any one of claims 1 to 5.

7. Use of the Cu2O-BSO antibacterial nanoparticles according to claim 6 in the preparation of a medicament for treating acute bacterial pneumonia, wherein the pathogenic bacteria causing acute bacterial pneumonia is methicillin-resistant Staphylococcus aureus.

8. A drug for treating acute bacterial pneumonia, characterized in that: The active ingredient is the Cu2O-BSO antibacterial nanoparticles according to claim 6, and the pathogenic bacteria causing acute bacterial pneumonia are methicillin-resistant Staphylococcus aureus.

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