Platelet antibacterial compound as well as preparation method and application thereof

Abstract

The invention provides a platelet antibacterial compound as well as a preparation method and application thereof. The platelet antibacterial compound comprises platelets and antibacterial substances, and the antibacterial substances are bacteriophages or antibiotics. The bacteriophage is coupled with platelets through a coupling agent to form bacteriophage-coupled platelets, and the antibiotics are endocytosed by the platelets to form the platelets loaded with the antibiotics. The cell structure of the platelets can help to mask the removal of antibacterial substances by the organism and improve the pharmacokinetics of the platelets. Besides, the platelets have the functions of naturally targeting inflammation and bacteria aggregation, so that targeted delivery of antibacterial substances can be realized, and the platelets can also intelligently respond to inflammation and bacteria signals to realize precise drug release. The invention provides technical support and a new thought for intelligent and accurate treatment of bacterial infection, and has a good clinical transformation prospect.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a platelet antibacterial complex, its preparation method, and its application. Background Technology

[0002] Lower respiratory tract infections (LRTIs) are a leading cause of morbidity and mortality worldwide, affecting 344 million people globally in 2021, including pneumonia and bronchiolitis. Bacteria, such as *Pseudomonas aeruginosa* and *Staphylococcus aureus*, are the most important pathogens in pneumonia. Currently, the main treatment for bacterial pneumonia in clinical practice is antibiotic therapy. However, due to antibiotic overuse, antimicrobial resistance (AMR) has become a serious challenge to global public health. According to a 2024 report by The Lancet, antimicrobial resistance is projected to directly cause more than 39 million deaths and 169 million related deaths between 2025 and 2050. Given the increasing concern about antibiotic-resistant bacteria and the lack of new antibiotics, more and more scientists are dedicated to developing new therapies to combat bacterial infections. Furthermore, systemic antibiotic therapy eliminates beneficial lung microbes and may lead to the persistence of pathogenic drug-resistant strains.

[0003] Bacteriophages are viruses that specifically infect bacteria, targeting and destroying host bacteria with high specificity while having little effect on other microorganisms. Phage therapy offers great hope for solving the growing crisis of antibiotic resistance, but it also faces many challenges. One problem hindering phage therapy is that bacteriophages, as foreign substances, are recognized by the body's immune system, remain in the bloodstream for a short time, and are rapidly cleared. Furthermore, as foreign viruses, bacteriophages lack specific targeting of organs and tissues within the body. Therefore, developing therapeutic phage therapies with organ targeting and longer blood circulation time is a key focus of phage clinical translational research.

[0004] Platelets are anucleate cell fragments released from megakaryocytes and are best known for their function in hemostasis. Approximately 75 billion platelets circulate in human blood, with an average lifespan of about 7 days, which can significantly improve the pharmacokinetics of intravenous therapies. Upon encountering endothelial injury or inflammation, platelets are rapidly recruited, including initial platelet binding and rolling, followed by platelet activation. Furthermore, in vitro and in experimental animal models, platelets actively target bacteria and aggregate at sites rich in bacterial-specific proteins, complement proteins C3a and C5a, and other signals from the acute phase of the infection response. Upon arrival at these sites, platelets scan the vascular surface to detect potential invaders and collect deposited bacteria. This microbial collection by migrating platelets enhances the activity of specialized phagocytes, and bacterial N-formyl peptides specifically bind to platelet formyl peptide receptors, leading to cytoskeletal rearrangement and calcium mobilization, promoting platelet activation and degranulation.

[0005] Therefore, engineered platelets carrying antibiotics or bacteriophages can be utilized, allowing the antibiotics or bacteriophages to actively migrate to the site of infection by leveraging the platelet's targeting of inflamed areas and bacteria. Once the engineered platelets reach the site of infection, environment-induced activation promotes the release of antimicrobial drugs and the killing of bacteria. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides a platelet antimicrobial complex, its preparation method, and its application. The platelet antimicrobial complex comprises platelets and an antimicrobial substance, wherein the antimicrobial substance is a bacteriophage or an antibiotic. The bacteriophage is coupled to platelets via a coupling agent to form phage-coupled platelets, and the antibiotic is endocytosed by platelets to form antibiotic-loaded platelets. The cellular structure of the platelet itself can help mask the body's clearance of the antimicrobial substance, improving its pharmacokinetics. Furthermore, platelets naturally target inflammation and bacterial aggregation, enabling targeted delivery of antimicrobial substances. Platelets can also intelligently respond to inflammatory and bacterial signals to achieve precise drug release. This invention provides technical support and new ideas for the intelligent and precise treatment of bacterial infections, and has good prospects for clinical translation.

[0007] On one hand, the present invention provides a platelet antimicrobial complex comprising platelets and an antimicrobial substance, wherein the platelets load the antimicrobial substance through endocytosis or are coupled with an antimicrobial substance through a coupling agent.

[0008] Furthermore, the antimicrobial substances include bacteriophages and antibiotics.

[0009] Furthermore, when the platelets load antibacterial substances via endocytosis, the antibacterial substances are antibiotics; when the platelets conjugate antibacterial substances via a conjugating agent, the antibacterial substances are bacteriophages.

[0010] The platelet antibacterial complex provided by this invention has two types: one is phage-coupled platelets, and the other is platelets loaded with antibiotics.

[0011] Bacteriophages can specifically target and destroy host bacteria with little effect on other microorganisms. However, two problems need to be addressed to hinder phage therapy: first, as foreign substances, phages are recognized by the body's immune system, remain in the bloodstream for a short time, and are quickly cleared; second, they lack specific targeting in organs and tissues, making it difficult to reach lesion areas. When the body encounters endothelial damage or inflammation, platelets are rapidly recruited and actively target and aggregate bacteria. Therefore, the phage-conjugated platelets provided in this invention combine the advantages of both phages and platelets, overcoming the disadvantages of phages being quickly cleared by the immune system and lacking specific targeting when used to eliminate pathogens. Currently, the main treatment for bacterial pneumonia in clinical practice is antibiotic therapy, but continuous use of antibiotics leads to drug resistance in pathogens. The phage-conjugated platelets provided in this invention offer a new method for the clinical treatment of bacterial pneumonia.

[0012] Although antibiotic therapy for bacterial pneumonia can lead to antibiotic resistance, it remains the mainstream treatment method. Injecting antibiotics into the body to treat bacterial pneumonia results in poor targeting of the antibiotics, leading to poor treatment efficacy. The antibiotic-loaded platelets provided by this invention can improve the targeting of antibiotics, thereby enhancing the therapeutic effect.

[0013] Furthermore, the coupling agent is tannic acid.

[0014] When bacteriophages are coupled to the platelet surface via tannins, this coupling method is non-covalent coupling. Compared with covalent coupling, non-covalent coupling is more conducive to the detachment and release of bacteriophages after platelet activation.

[0015] In some methods, phage-conjugated platelets (PPCs) were prepared using non-covalently coupled reagent tannic acid and traditional covalently coupled reagents SMCC and streptavidin-biotin systems, respectively. The in vitro antibacterial activity, blood retention, and targeted enrichment at infection sites of phage-conjugated platelets prepared by different methods were compared. The results showed that PPCs prepared by tannic acid had stronger in vitro antibacterial activity, longer retention time in blood circulation, and stronger ability to target lung lesions.

[0016] Furthermore, the bacteriophage includes one or more bacteriophages capable of infecting Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae, and Acinetobacter baumannii.

[0017] Furthermore, the antibiotics include one or more of the following: penicillins, cephalosporins, macrolides, aminoglycosides, tetracyclines, quinolones, sulfonamides, and glycopeptides.

[0018] The penicillins mentioned include, but are not limited to, penicillin G, amoxicillin, etc.

[0019] The cephalosporins include, but are not limited to, cefuroxime and ceftriaxone.

[0020] The macrolides include, but are not limited to, azithromycin and erythromycin.

[0021] The aminoglycosides include, but are not limited to, gentamicin and amikacin.

[0022] The tetracyclines include, but are not limited to, doxycycline, minocycline, etc.

[0023] The quinolones include, but are not limited to, levofloxacin and ciprofloxacin.

[0024] On the other hand, the present invention provides a method for preparing the platelet antibacterial complex as described above, comprising the following steps: (1) Platelets are extracted from the blood and prostaglandin E1 is added to the platelets; (2) When the antibacterial substance is a bacteriophage, the bacteriophage is extracted from the Pseudomonas aeruginosa strain, and the bacteriophage is mixed with tannic acid to obtain bacteriophage nanoparticles; the bacteriophage nanoparticles are incubated with the platelets from step (1) to obtain bacteriophage-coupled platelets; (3) When the antibacterial substance is an antibiotic, the antibiotic is incubated with the platelets from step (1) to obtain platelets loaded with antibiotics.

[0025] In the above-mentioned preparation of platelet antibacterial complexes, the role of adding prostaglandin E1 is to maintain the resting state of platelets in vitro, prevent premature activation and aggregation, and ensure the effective coupling of platelets with phage nanoparticles and the endocytosis of antibiotics. Prostaglandin E1 can prevent platelets from being activated during in vitro extraction and processing by increasing the intracellular cAMP level in platelets (targeting only the platelet-inhibiting activation pathway without affecting its targeting function), thereby preventing them from adhering, aggregating, and releasing their contents. This preserves the natural chemotaxis of platelets to lung infection foci, laying the foundation for targeted delivery of antibacterial substances, while also reducing platelet loss and improving the success rate of incubation with phages / antibiotics. Therefore, the addition of prostaglandin E1 can ensure that phage-coupled platelets and antibiotic-loaded platelets remain in an inactivated state in vitro.

[0026] In existing technologies, the methods for constructing targeted delivery systems by combining antibiotics with platelets mainly fall into three categories: The first involves encapsulating antibiotics with lipid nanoparticles and then conjugating them to platelets. This method is not only cumbersome and involves many steps, but also requires the introduction of exogenous lipid carriers, potentially increasing the risk of platelet immunogenicity. Furthermore, the conjugation process between nanoparticles and platelets easily damages the platelet membrane surface structure, leading to impaired natural physiological activity. The second method involves platelet electroporation to encapsulate antibiotics. This method relies on a high-voltage electric field to physically perforate the platelet membrane to allow antibiotic entry. This process directly damages the integrity of the platelet membrane, easily triggering platelet activation, leakage of contents, or functional impairment. Firstly, electroporation equipment is complex to operate and requires strict control of process parameters, making it difficult to meet the needs of large-scale preparation, and the equipment cost is high. Secondly, antibiotics are covalently coupled to the platelet surface through coupling agents. This approach requires the use of chemical coupling agents (such as cross-linking agents, activators, etc.) to mediate the covalent binding of antibiotics to platelet surface proteins. On the one hand, chemical coupling agents may have toxic effects on platelets. On the other hand, the coupling reaction can easily modify the target-related proteins on the platelet surface (such as integrins, selectins, etc.), causing platelets to lose their natural chemotactic targeting ability to lung infection foci. Furthermore, the introduction of exogenous coupling agents requires additional safety assessments, increasing the resistance to clinical translation.

[0027] This invention employs a platelet endocytosis method to prepare antibiotics, which does not rely on exogenous carriers, chemical conjugates, or physical perforation. This minimizes the risk of damage to platelet membrane structure, membrane protein activity, and intracellular function caused by chemical / physical factors, ensuring that platelets maintain good physiological stability after antibiotic loading. Secondly, because no modification is made to platelet surface targeting-related proteins, antibiotic-loaded platelets can still accurately recognize inflammatory signals from lung infection foci, maintaining their inherent targeting and homing capabilities. Furthermore, the method has a simplified operation process, making it easy to standardize and scale up production. Simultaneously, because no exogenous excipients are introduced, the preparation process is more in line with existing regulatory standards for clinical platelet processing, reducing unknown risks during regulatory review and lowering resistance to clinical translation. In addition, this approach has low raw material and production costs, making it more promising for industrial application.

[0028] Further, the concentration of prostaglandin E1 in step (1) is 1-10 mM; the concentration of the bacteriophage in step (2) is 10 mM. 8 -10 11 The concentration of PFU / mL, the concentration of tannic acid is 0.1-20 mg / mL, the particle size of the phage nanoparticles is 50-500 nm; the concentration of the antibiotic in step (3) is 10-1000 μg / mL.

[0029] In another aspect, the present invention provides the use of the platelet antibacterial complex as described above in the preparation of a medicament for treating bacterial pneumonia.

[0030] In some embodiments, the phage-conjugated platelets prepared according to this invention exhibit an inhibition rate of over 70% against the formation of immature bacterial biofilms and a destruction rate of up to 60% against mature bacterial biofilms, demonstrating a significant advantage in combating biofilm infections. Bacterial biofilms are a core cause of chronic infections and antibiotic resistance: biofilms form a dense extracellular matrix barrier, hindering antibiotic penetration; simultaneously, they slow bacterial metabolism, significantly reducing the killing efficiency of antibiotics, leading to the ineffectiveness of conventional antibacterial treatments (such as antibiotics), and resulting in chronic and recurrent infections. Therefore, the phage-conjugated platelets prepared according to this invention provide a core prerequisite for a novel treatment strategy for antibiotic-resistant infections.

[0031] In some embodiments, the phage-conjugated platelets prepared according to the present invention have excellent effects in treating both bacterial and drug-resistant bacterial pneumonia, can efficiently clear pathogens from the lungs, and have low levels of lung inflammation, effectively reducing inflammatory damage to the lungs.

[0032] In some methods, direct intravenous injection of antibiotics (ciprofloxacin) into bacterial pneumonia model mice has limited efficacy in treating pneumonia, with only 10% of mice surviving after 4 days of treatment. However, injection of the antibiotic-loaded platelets provided by this invention resulted in 80% of mice surviving after 5 days of treatment, significantly prolonging the survival time of bacterial pneumonia mice. This indicates that the antibiotic-loaded platelets can more efficiently deliver ciprofloxacin to the site of infection and exert excellent therapeutic effects.

[0033] In another aspect, the present invention provides the use of a composition for preparing phage-conjugated platelets, said composition being prostaglandin E1 and tannic acid.

[0034] The beneficial effects of this invention include: 1. The platelet antibacterial complex provided by the present invention has two types: one is phage-coupled platelets, and the other is platelets loaded with antibiotics. 2. The phage-conjugated platelets have a long retention time in the hematological circulation and a strong ability to target the site of infection. 3. The phage-conjugated platelets have an inhibition rate of over 70% against the formation of immature bacterial biofilms and a destruction rate of up to 60% against mature bacterial biofilms, demonstrating outstanding advantages in anti-biofilm infection and providing a core premise for a new treatment strategy for antibiotic-resistant infections. 4. The phage-conjugated platelets have excellent effects in treating both bacterial pneumonia and drug-resistant bacterial pneumonia. They can effectively clear pathogens from the lungs and reduce lung inflammation levels, thus effectively reducing inflammatory damage to the lungs. 5. The antibiotic-loaded platelets can improve the targeting of antibiotics to the lesion area, thereby improving the therapeutic effect of antibiotics; 6. The present invention provides a composition for preparing phage-conjugated platelets, comprising prostaglandin E1 and tannic acid. The phage-conjugated platelets prepared by this composition have stronger in vitro antibacterial activity, longer retention time in blood circulation, and stronger ability to target lung lesions. Attached Figure Description

[0035] Figure 1 The process for preparing phage-conjugated platelets (PPCs) for Example 1; Figure 2 a represents the hydrodynamic diameter of the PPC determined in Example 1; Figure 2 b is a confocal microscope image of the PPC in Example 1, scale bar, 5 μm; Figure 2 c is a scanning electron microscope image of PPC (left) and platelets (right) in Example 1, scale bar, 500 nm; Figure 2 d represents the phage plaque generated by PPC on an agar plate in Example 2; Figure 2 e represents the number of phages released by the PPC in Example 2; Figure 2 f is a confocal microscope image of the binding of bacteriophage and bacteria in Example 2, scale bar, 5 μm; Figure 2 g represents the in vitro antibacterial curve of PPC in Example 2; Figure 2 h represents the blood retention time of the PPC in Example 2; Figure 2 i represents the enrichment of phages in the lungs of mice after intravenous injection of PPC in Example 2; Figure 2 j represents the quantitative analysis of the fluorescence intensity of bacteriophages in the lungs in Example 2; Figure 3 a is a photograph of immature biofilms after different treatments with crystal violet staining in Example 3; Figure 3 b represents the absorbance of the crystal violet eluent of immature biofilms after different treatments in Example 3 at 570 nm. Figure 3 c represents the inhibition rate of immature biofilms after different treatments in Example 3; Figure 3 d shows confocal images and corresponding 3D images of immature biofilms stained with DMAO (green) after different treatments in Example 3. Scale bar: 50. μ m; Figure 3 e shows scanning electron microscope images of immature biofilms after different treatments following gradient dehydration in Example 3. (Scale bar: 5) μ m; Figure 3 f is a photograph of mature biofilms after different treatments with crystal violet staining in Example 3; Figure 3 g represents the absorbance of the crystal violet eluent of mature biofilms after different treatments in Example 3 at 570 nm. Figure 3 h represents the damage rate of the mature biofilm after different treatments in Example 3; Figure 3 i represents confocal images and corresponding 3D images of DMAO-stained (green) mature biofilms after different treatments in Example 3. Scale bar: 50. μ m; Figure 3 j is a scanning electron microscope image of the mature biofilm after different treatments following gradient dehydration in Example 3. Scale bar: 5 μ m; Figure 4 Example 4 illustrates the process of PPC treatment for bacterial pneumonia; Figure 5 a is a schematic diagram of the experiment in Example 4 on the treatment of bacterial pneumonia in mice with PPC; Figure 5 b represents the survival rate of pneumonia mice treated with PBS, bacteriophages, bacteriophage nanoparticles, and PPC in Example 4; Figure 5 c represents the quantitative measurement of phage load in the lungs of pneumonia mice after treatment in Example 4; Figure 5 d represents the quantitative measurement of bacterial load in the lungs of pneumonia mice after treatment in Example 4; Figure 5 e represents the pathological staining results of lung tissue after treatment in pneumonia mice in Example 4; Figure 5 f represents the lung damage score in mice with pneumonia after treatment in Example 4; Figure 5 gi is a quantitative analysis of the levels of IL-1β, TNF-α and IL-6 in lung tissue homogenates of pneumonia mice 12 and 24 hours after treatment in Example 4; Figure 6 a shows the growth curves of different concentrations of ciprofloxacin against Pseudomonas aeruginosa in Example 4; Figure 6 b shows the in vitro antibacterial curve of PPC against drug-resistant bacteria in Example 4; Figure 6 c is a schematic diagram of the experiment in Example 4 on the treatment of mice with drug-resistant bacterial pneumonia using PPC; Figure 6 d represents the survival rate of mice with drug-resistant bacterial pneumonia treated with PBS, bacteriophages, bacteriophage nanoparticles, and PPC in Example 4. Figure 6 For example, in Example 4, quantitative analysis of IL-1β, TNF-α and IL-6 levels in lung tissue homogenates of mice with drug-resistant bacterial pneumonia at 12 and 24 hours after treatment; Figure 6 h represents the pathological staining results of lung tissue after treatment in mice with drug-resistant bacterial pneumonia, as described in Example 4. Figure 6 i represents the lung damage score after treatment of mice with drug-resistant bacterial pneumonia in Example 4; Figure 7 a represents the platelet loading in ciprofloxacin solutions of different concentrations in Example 6; Figure 7 b is the drug release curve of Cip-PLT in Example 6; Figure 7 c represents the survival curve of mice treated with Cip-PLT for bacterial pneumonia in Example 6. Detailed Implementation

[0036] The present invention will be further described in detail below with reference to the embodiments. It should be noted that the embodiments described below are intended to facilitate the understanding of the present invention and do not limit it in any way.

[0037] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0038] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0039] Example 1: Preparation of engineered platelet complex 1. Platelet extraction and purification Fresh mouse blood was collected in test tubes containing acidic citrate glucose anticoagulant solution at a ratio of 1:(9-20) to plasma. The plasma was centrifuged twice to obtain purified platelets. The purified platelets were resuspended in PBS and 1-10 mM prostaglandin E1 was added.

[0040] 2. Preparation of phage-conjugated platelets (PPCs) like Figure 1 The image shows the process for preparing phage-conjugated platelets.

[0041] Take the supernatant of hospital sewage and LB medium at a ratio of 1:(1-10) and mix them gently by shaking. Add 10-1000 μL of Pseudomonas aeruginosa strain in the logarithmic growth phase. Incubate the mixture in a shaking incubator at 22-38℃ and 100-300 rpm for 8-16 h. Centrifuge at 2000-8000 g for 5-30 min, collect the supernatant, filter, and collect the filtrate to obtain the bacterial lysate containing the target bacteriophage. The concentration of the bacteriophage is 10. 8 -10 11 PFU / mL.

[0042] Bacteriophages were added dropwise to a 0.1-20 mg / mL tannic acid aqueous solution and shaken for 1-30 min to prepare phage nanoparticles (Ta-Phage).

[0043] The obtained phage nanoparticles were incubated with platelets for 10-300 min to obtain phage-conjugated platelets (PPCs).

[0044] 3. Preparation of antibiotic-loaded platelets The purified platelets were incubated with 10-1000 μg / mL antibiotic solution (pH 5.5-8.5) for 10-300 min, centrifuged at 50-300g for 5-30 min to remove free antibiotics and obtain antibiotic-loaded platelets.

[0045] Furthermore, the phage-conjugated platelets (PPCs) prepared above were characterized. First, the hydrodynamic diameter and distribution of the PPCs were determined using dynamic light scattering (DLS), and the results are as follows: Figure 2 As shown in Figure a, the particle size of PPC ranges from 50 to 500 nm, with the peak particle size distribution concentrated at approximately 100 nm, indicating that the prepared PPC particles are uniform in size. This size is beneficial for diffusion, cell-cell interaction, and targeted delivery within the biological system (too small particles are easily cleared, while too large particles may trigger immune responses or abnormal tissue retention). Next, platelets in the PPC were labeled with FITC-labeled CD41 antibody (green, CD41 is a marker glycoprotein on the platelet surface), and bacteriophages were tracked using Cy5 (red) fluorescence. Detection was then performed using a confocal laser scanning microscope. The confocal microscope image is shown below. Figure 2 As shown in b, the green (platelet) and red (phage) signals co-localize in the Merge plot, directly proving that the phage was successfully coupled to the platelet surface. Subsequently, the surface morphology of PPCs and platelets was observed by scanning electron microscopy (SEM), as shown in the SEM images. Figure 2As shown in Figure c, the SEM image of PPC on the left shows granular structures (phage nanocomplexes) attached to the platelet surface, while the platelet surface on the right without phage conjugation is relatively smooth. This indicates that phage conjugation significantly alters the surface morphology of the platelets, providing structural evidence of successful conjugation.

[0046] In this embodiment, the bacteriophage selected is the bacteriophage that infects Pseudomonas aeruginosa. In addition, bacteriophages that infect Staphylococcus aureus, Klebsiella pneumoniae and Acinetobacter baumannii can also be selected, and there is no significant difference in the PPCs made from them.

[0047] Example 2: Pharmacokinetics and efficacy validation of phage-conjugated platelets (PPC) This embodiment uses the PPC prepared in Example 1 as the experimental object to test its bioactivity, in vitro antibacterial ability, phage release pattern, retention characteristics in blood circulation, and in vivo targeted enrichment ability at the site of infection.

[0048] 1. Bioactivity and in vitro antibacterial ability of PPC The host bacterium (Pseudomonas aeruginosa) was labeled with blue fluorescent dye, and the bacteriophages in the PPC were labeled with red fluorescent dye. After mixing the PPC with the host bacterium (Pseudomonas aeruginosa), the binding of the two was detected using a confocal laser scanning microscope. The detection results are as follows: Figure 2 As shown in f, the red (phage) and blue (host bacteria) signals are co-located in the Merge plot, proving that phages in PPC can effectively bind to the surface of target bacteria.

[0049] Furthermore, the bioactivity of PPC was assessed using a plaque formation assay. PPC was mixed with host bacteria (Pseudomonas aeruginosa) and plated; phage lysis of the bacteria resulted in the formation of transparent plaques. Plaques, such as… Figure 2 As shown in d, it is demonstrated that the phages released by PPC still have biological activity and can effectively infect and lyse target bacteria. The coupling process did not destroy the antibacterial function of the phages.

[0050] Furthermore, PPC, free phage, and Ta-Phage (phage nanoparticles prepared in Example 1) were co-cultured with host bacteria (Pseudomonas aeruginosa), with host bacteria cultured alone serving as a blank control. During the culture process, the absorbance of the bacterial solution at 600 nm was measured every 2 hours, and a bacterial growth curve was plotted for 0-12 hours. The bacterial growth curve is shown below. Figure 2As shown in g, in the first 4 hours, the bacterial growth in the PPC group, the free phage group, and the Ta-Phage group was not significantly different from that in the control group. After 4 hours, the bacteria in the PPC group, the free phage group, and the Ta-Phage group did not amplify, while the bacteria in the control group amplified. This indicates that the ability of the phage in the PPC group to infect the host bacteria was not significantly different from that of the free phage.

[0051] 2. The release pattern of bacteriophages in PPC and the retention characteristics of PPC in blood circulation. In Example 1, because prostaglandin E1 was added to the platelets, the prepared PPC was an unactivated platelet-conjugated PPC. Two groups of unactivated platelet-conjugated PPCs prepared in Example 1 at the same concentration were co-cultured with host bacteria (Pseudomonas aeruginosa), with thrombin (to activate platelets) added to one group. Bacterial culture was collected at 1, 2, 4, 8, and 16 hours of co-culture, and the amount of phage released from the PPC was counted using plaque counting. The results of the number of phages released from the PPC are as follows: Figure 2 As shown in Figure e, both groups of PPCs released phages over time. The activated platelet group (with added thrombin) released more and faster phages, while the inactivated platelet group (without added thrombin) released phages in a slow-release manner. This indicates that the activation state of platelets regulates the phage release kinetics. Thrombin is often present at sites of infection or inflammation; therefore, this characteristic allows the inactivated platelet-conjugated PPCs to accumulate at the target site in vivo before being released efficiently.

[0052] The PPC, free phage, and Ta-phage prepared in Example 1 were intravenously injected into mice. Blood samples were collected from the mice at 1, 3, 6, and 9 hours post-injection. The phage content was quantified by plaque counting, and concentration-time curves were plotted. The concentration-time curves are shown below. Figure 2 As shown in h, the phage content in the blood of all three groups gradually decreased over time. However, at the 9th hour, the phage content in the blood of the PPC group was significantly higher than that of the other two groups. This indicates that the phage in the PPC group remained in the blood for a significantly longer time than that in the free phage group. This suggests that platelets, as a carrier, can prolong the blood circulation time of phages, reduce the possibility of them being rapidly cleared (such as by renal filtration or immune phagocytosis), and facilitate their delivery throughout the body.

[0053] 3. PPC's ability to target and accumulate at infection sites in vivo. Fluorescently labeled PPC, free phage, and Ta-Phage were intravenously injected into pneumonia model mice, respectively. Fluorescently labeled PPC was intravenously injected into healthy mice. The enrichment of phage in the lungs of each mouse was detected by fluorescence imaging at 1, 2, and 4 hours post-injection. Fluorescence imaging of the lungs of each mouse is shown below. Figure 2As shown in Figure i, the lung fluorescence intensity of pneumonia model mice injected with PPC was significantly higher than that of pneumonia model mice injected with Phage, Ta-Phage, and healthy mice injected with PPC. Quantitative results of lung fluorescence intensity for each mouse type are shown below. Figure 2 As shown in j, consistent with the fluorescence results, this indicates that platelets enhance the lung targeting of PPC, and that PPC only targets lung lesion areas and does not target uninfected lungs.

[0054] Example 3: Inhibition and disruption of bacterial biofilm formation by PPC Bacterial biofilms are a core contributing factor to chronic infections and antibiotic resistance: biofilms form a dense extracellular matrix barrier, hindering antibiotic penetration; they also slow down bacterial metabolism, significantly reducing the effectiveness of antibiotics in killing bacteria, leading to the ineffectiveness of conventional antibacterial treatments (such as antibiotics), and resulting in chronic and recurrent infections. Therefore, evaluating the anti-biofilm ability of the PPC prepared in Example 1 is a crucial prerequisite for verifying whether it can provide a novel treatment strategy for antibiotic-resistant infections.

[0055] 1. Inhibition experiment of PPC on immature biofilms Bacteria (Pseudomonas aeruginosa) were inoculated into the culture system and cultured until the biofilm was immature (bacteria began to aggregate but the matrix was not fully formed); the bacteria were treated with different methods: Control (blank control), Cip (ciprofloxacin), PLT (platelets), Phage (free phage), Ta-Phage (phage nanoparticles prepared in Example 1), and PPC (phage-conjugated platelets prepared in Example 1).

[0056] Each group of bacteria was stained with crystal violet (biofilms can adsorb crystal violet, and the intensity of the color reflects the amount of biofilm), and the degree of staining was observed; crystal violet was eluted with ethanol, and the absorbance (OD) of the eluent at 570 nm was measured. 570 And calculate the inhibition rate, inhibition rate = (Control group OD - Experimental group OD) / Control group OD × 100%.

[0057] Crystal violet staining results as follows Figure 3 As shown in Figure a, the Control group stained the darkest (with the most biofilm), while the PPC group stained the lightest (with the least biofilm). The absorbance of the crystal violet eluent at 570 nm is shown in Figure a. Figure 3 As shown in b, the inhibition rate is as follows: Figure 3 As shown in c, it can be seen that PPC has a significantly higher inhibition rate on immature biofilms than other groups, with an inhibition rate of over 70%; Cip has a very low inhibition rate on immature biofilms, with an inhibition rate of less than 10%.

[0058] Furthermore, each group of biofilms was labeled with DMAO fluorescent dye (green), and 2D fluorescence images and 3D reconstructed images were captured using a laser confocal microscope (CLSM) to observe the spatial structure of the biofilms, including thickness and distribution. Figure 3 As shown in d, the green fluorescence of the biofilm in the Control group was dense and the 3D structure was thick, while the fluorescence of the PPC group was sparse and the 3D structure was thin. This indicates that PPC inhibited the formation of the spatial structure of the biofilm, while the other groups were not as effective as PPC in inhibiting the formation of the biofilm.

[0059] Furthermore, the biofilms after each treatment were subjected to gradient dehydration, and the microstructure of bacterial aggregation and biofilm matrix was observed using scanning electron microscopy (SEM). Figure 3 As shown in Figure e, the Control group showed dense bacterial aggregation and biofilm matrix, while the PPC group showed dispersed bacteria and incomplete biofilm structure. This demonstrates from the ultrastructural level that PPC inhibits biofilm formation, while the other groups were less effective at inhibiting biofilm formation than PPC.

[0060] 2. Experiment on the disruption of mature biofilms by PPC Bacteria (Pseudomonas aeruginosa) were inoculated into the culture system and cultured until the biofilm matured (the biofilm matrix was intact and the structure was dense). The bacteria were then treated with different methods: Control (blank control), Cip (ciprofloxacin), PLT (platelets), Phage (free phage), Ta-Phage (phage nanoparticles prepared in Example 1), and PPC (phage-conjugated platelets prepared in Example 1).

[0061] Each group of bacteria was stained with crystal violet (biofilms can adsorb crystal violet, and the intensity of the color reflects the amount of biofilm), and the degree of staining was observed; crystal violet was eluted with ethanol, and the absorbance (OD) of the eluent at 570 nm was measured. 570 And calculate the destruction rate, destruction rate = (Control group OD - Experimental group OD) / Control group OD × 100%.

[0062] Crystal violet staining results as follows Figure 3 As shown in f, the Control group stained the darkest (with the most biofilm residue), while the PPC group stained the lightest (with the least biofilm residue). The absorbance of the crystal violet eluent at 570 nm is shown in f. Figure 3 As shown in g, the destruction rate is as follows: Figure 3 As shown in h, it can be seen that PPC caused significantly higher damage to mature biofilms than other groups, with a damage rate as high as 60%; CIP and PLT caused very little damage to mature biofilms, with a damage rate of less than 10%.

[0063] Furthermore, each group of biofilms was labeled with DMAO fluorescent dye (green), and 2D fluorescence images and 3D reconstructed images were captured using a laser confocal microscope (CLSM) to observe the spatial structure of the biofilms, including thickness and distribution. Figure 3 As shown in i, the mature biofilm in the Control group exhibited strong green fluorescence and a dense 3D structure, while the PPC group showed weak fluorescence and a loose and fragmented 3D structure. This demonstrates that PPC disrupted the spatial structure of the mature biofilm. The other groups were less effective at disrupting the biofilm than PPC. Among them, the CIP and PLT groups showed very little difference in the density of green fluorescence and 3D structure compared to the Control group, indicating that CIP and PLT had a very low rate of disruption on the mature biofilm.

[0064] Furthermore, the biofilms after each treatment were subjected to gradient dehydration, and the microstructure of bacterial aggregation and biofilm matrix was observed using scanning electron microscopy (SEM). Figure 3 As shown in j, the mature biofilm structure in the Control group was intact and the bacteria were encapsulated in the matrix. In the PPC group, the biofilm matrix was ruptured and the bacteria were exposed or dispersed. This demonstrates from the ultrastructural level that PPC disrupts biofilm formation, while the other groups were not as effective as PPC in disrupting biofilm.

[0065] The above experiments on the inhibition of immature biofilms and the destruction of mature biofilms by PPC show that PPC is significantly better than antibiotics (Cip), platelets (PLT), free phages (Phage), or phage nanoparticles (Ta-Phage) prepared in Example 1, indicating that it has outstanding advantages in anti-biofilm infection.

[0066] Example 4: PPC treatment for bacterial pneumonia The process of preparing PPC for treating bacterial pneumonia in Example 1 is as follows: Figure 4 As shown: Bacterial infection in the lungs leads to pneumonia, which in turn causes inflammation in the lung tissue. After PPC enters the lung tissue, it is stimulated by the inflammation signals, which activates platelets. The activated platelets generate platelet microparticles (PMPs) and promote the release of bacteriophages to infect pathogenic bacteria. At the same time, platelet microparticles can aggregate pathogenic bacteria, allowing the released bacteriophages to contact the bacteria more efficiently and further enhancing the antibacterial effect.

[0067] 1. PPC is used for the treatment of antibacterial pneumonia. like Figure 5Figure a shows the experimental procedure. A bacterial pneumonia model in mice was established by intratracheal inoculation with bacteria. Two hours after modeling, different treatments were administered intravenously: the control group was injected with PBS (blank buffer), while the experimental groups were injected with Phage (free bacteriophage), Ta-Phage (phage nanoparticles prepared in Example 1), and PPC (phage-conjugated platelets prepared in Example 1), respectively. Long-term monitoring and multi-index analysis were then carried out.

[0068] Mice in each group were observed for 14 consecutive days, and the number of surviving mice was recorded daily to plot survival curves. Figure 5 As shown in b, the survival rate of mice in the PBS group (blank control) decreased rapidly, and all mice died by day 3. The survival rates of mice in the Phage and Ta-Phage groups improved to some extent, but remained at a low level. From day 4 to 14, mice in the Phage group maintained a survival rate of 20%, and mice in the Ta-Phage group maintained a survival rate of 30%. The survival rate of mice in the PPC group was significantly higher than that of other groups, and the long-term survival rate was even higher. From day 2 to 14, mice maintained a survival rate of 90%, which proves that PPC can effectively improve the survival probability of mice with bacterial pneumonia.

[0069] On the second day of treatment, lung tissue was collected from surviving mice in each group. A portion of the lung tissue was fixed, sectioned, and stained with hematoxylin and eosin (HE) for microscopic observation of pathological changes. The lung morphology was observed through histopathological staining. The other portion of the lung tissue was homogenized. One portion of the homogenate was used to determine the phage load in the lung tissue (PFU / g tissue) using plaque counting. The other portion of the homogenate was serially diluted and spread onto bacterial culture medium. After incubation, colony-forming units were counted to quantify the residual bacterial load in the lungs (CFU / g tissue).

[0070] Phage load in lung tissue of mice in each group as follows Figure 5 As shown in c, the residual bacterial load in the lung tissue is as follows: Figure 5 As shown in d, the phage load in the lungs of the PPC group was significantly higher than that of the Phage and Ta-Phage groups, indicating that platelets, as a carrier, can enhance the targeted delivery efficiency of phages to lung infection lesions, resulting in a higher local phage concentration. Therefore, bacteria in the lungs of the PPC group can be cleared more efficiently.

[0071] Histopathological staining results as follows Figure 5 As shown in Figure e, the PBS group showed severe inflammatory infiltration and structural disorder in the lung tissue; the PPC group showed fewer inflammatory cells in the lung tissue, directly demonstrating the therapeutic effect of PPC on bacterial pneumonia.

[0072] Lung tissues from surviving mice in each group were collected at 12 and 24 hours after treatment, homogenized, and the levels of pro-inflammatory cytokines IL-1β, TNF-α, and IL-6 were quantified using enzyme-linked immunosorbent assay (ELISA). These factors are core biomarkers of lung inflammation. The detection results of pro-inflammatory cytokines are as follows: Figure 5 As shown in the figure, the levels of these three cytokines in the PBS group were highest at 12 and 24 hours (indicating the most severe inflammation, with continuous bacterial stimulation leading to excessive inflammation); the levels of these three cytokines in the PPC group were lowest at both 12 and 24 hours, indicating that PPC can not only clear bacteria but also effectively reduce lung inflammation and decrease infection-induced inflammatory damage; the levels of these three cytokines in the Phage and Ta-Phage groups were lower than those in the control group, but still higher than those in the PPC group.

[0073] Furthermore, the lung damage in mice treated with different groups was scored, and the scoring results are as follows: Figure 5 As shown in f, lung damage is minimized after treatment with PPC for bacterial pneumonia.

[0074] 2. PPC treatment for drug-resistant bacterial pneumonia First, *Pseudomonas aeruginosa* was inoculated into culture media containing different concentrations of ciprofloxacin (5, 10, 20, 40, 80 μg / mL), with a control group (no drug) serving as the general control. The OD (octane rating) of the culture medium was measured at 0, 2, 4, 6, 8, 10, and 12 h. 600 Draw a growth curve. The growth curve is as follows: Figure 6 As shown in Figure a, the higher the concentration of ciprofloxacin, the more significant the inhibition of Pseudomonas aeruginosa growth. When the concentration of ciprofloxacin is between 5-40 μg / mL, the inhibition of Pseudomonas aeruginosa growth is not significant. Only when the concentration of ciprofloxacin is increased to 80 μg / mL is the growth of Pseudomonas aeruginosa inhibited. 600 The effect is almost negligible. Therefore, *Pseudomonas aeruginosa* is resistant to low concentrations of ciprofloxacin.

[0075] Furthermore, drug-resistant Pseudomonas aeruginosa was inoculated into LB medium containing PPC, Cip (ciprofloxacin), Ta-Phage, and Phage, while the control group was LB medium without any added substances. OD values ​​were measured at 0, 2, 4, 6, 8, 10, and 12 h. 600 Draw a growth curve. The growth curve is as follows: Figure 6 As shown in b, the OD values ​​of the PPC group, Ta-Phage group, and Phage group hardly increased, indicating that they had a significant inhibitory effect on drug-resistant bacteria in vitro; the OD value of the Cip group (ciprofloxacin) increased significantly, similar to the trend of the control group, indicating that the bacteria were resistant to ciprofloxacin.

[0076] Furthermore, PPC was used to treat drug-resistant bacterial pneumonia in mice, and the experimental procedure was as follows: Figure 6 As shown in c. A mouse model of drug-resistant bacterial pneumonia was established by intratracheal inoculation with drug-resistant Pseudomonas aeruginosa. Two hours after modeling, different treatments were administered intravenously: the control group was injected with PBS (blank buffer), while the experimental groups were injected with Phage (free bacteriophage), Ta-Phage (phage nanoparticles), Cip (ciprofloxacin), and PPC (phage-coupled platelets), respectively. Long-term monitoring and multi-index analysis were then performed.

[0077] Mice in each group were observed for 14 consecutive days, and the number of surviving mice was recorded daily to plot survival curves. Figure 6 As shown in d, the survival rate of mice in the PBS group (blank control) decreased rapidly, and all mice died by day 8. The survival rate of mice in the Cip group was improved compared with the control group, but only 10% of mice survived from day 4 to day 14. The survival rates of mice in the Phage and Ta-Phage groups were further improved compared with the Cip group, but were still at a low level. From day 4 to day 14, mice in the Phage group maintained a survival rate of 30%, and mice in the Ta-Phage group maintained a survival rate of 40%. The survival rate of mice in the PPC group was significantly higher than that of other groups, and the long-term survival rate was higher. The mice maintained a survival rate of 80% from day 2 to day 14, which proves that PPC can significantly improve the survival rate of mice with drug-resistant bacterial pneumonia.

[0078] Lung tissues were collected from surviving mice at 12 and 24 hours after treatment. A portion of the lung tissue was homogenized, and the levels of pro-inflammatory cytokines IL-1β, TNF-α, and IL-6 were quantified using enzyme-linked immunosorbent assay (ELISA). These factors are core markers of pulmonary inflammation. The other portion of lung tissue was fixed, sectioned, and stained with hematoxylin and eosin (HE). Pathological changes were observed under a microscope, and lung morphology was examined through histopathological staining.

[0079] The results of the detection of pro-inflammatory cytokines are as follows: Figure 6 As shown in the figure, the levels of these three cytokines in the PBS group were highest at 12 and 24 hours (when the inflammatory response was most severe); the levels of these three cytokines in the PPC group were lowest at both 12 and 24 hours, indicating that PPC can not only clear drug-resistant bacteria, but also effectively reduce lung inflammation and inhibit the release of pro-inflammatory cytokines; the levels of these three cytokines in the Phage, Ta-Phage, and Cip groups were lower than those in the control group, but still higher than those in the PPC group.

[0080] The pathological staining results of lung tissue 24 hours after treatment in each group are as follows: Figure 6As shown in h, the PBS group exhibited extensive inflammatory cell infiltration and severe alveolar structure destruction in the lung tissue; the Phage, Ta-Phage, and Cip groups still showed significant lung tissue damage; the PPC group showed the least inflammatory infiltration and the alveolar structure was closest to normal. This indicates that PPC can effectively reduce lung pathological damage caused by drug-resistant bacteria.

[0081] Furthermore, lung tissue damage was scored in different treatment groups. The score distribution is as follows: Figure 6 As shown in i, the PBS group had the highest injury score, while the PPC group had a significantly lower score than the other groups, demonstrating that PPC treatment resulted in the mildest lung damage after pneumonia caused by drug-resistant bacteria.

[0082] Example 5: Screening of phage-platelet coupling mechanisms in PPC In Example 1, bacteriophages were coupled to the platelet surface via the coupling reagent tannic acid. This example changes the method of coupling bacteriophages to the platelet surface. Other methods include coupling with the coupling reagent SMCC and coupling with the streptavidin-biotin system.

[0083] PPC prepared by tannic acid is denoted as Ta-PPC, PPC prepared by SMCC is denoted as SMCC-PPC, and PPC prepared by the streptavidin-biotin system is denoted as SA-Biotin-PPC.

[0084] The number of PPC-conjugated phages prepared by the three different conjugation methods was determined. Ta-PPC, SMCC-PPC, and SA-Biotin-PPC were co-cultured with host bacteria (Pseudomonas aeruginosa) on double-layer agar plates, with host bacteria cultured alone serving as a blank control. After 12 hours of incubation, the formation of phage plaques was observed and counted. The results are shown in Table 1 below.

[0085] Table 1. Number of PPC-conjugated phages prepared by different conjugation methods

[0086] According to the data in Table 1, Ta-PPC can achieve an average of about 17 phages conjugated per platelet, while SMCC-PPC and SA-Biotin-PPC can hardly conjugate any phages. Therefore, PPC prepared via tannic acid is preferred.

[0087] Example 6: In vitro evaluation of antibiotic-loaded platelets and their efficacy in treating bacterial pneumonia. Platelets loaded with antibiotics were prepared according to the method in Example 1, with Cip (ciprofloxacin) as an example of antibiotic, and Cip-loaded platelets (Cip-PLT) were prepared.

[0088] 1. In vitro evaluation of Cip-PLT First, the effect of different concentrations of Cip solution on platelet drug loading was investigated. Platelets were co-incubated with Cip solution at concentrations of 50, 100, 200, and 400 μg / mL, and the final drug loading on the platelets was then measured. Figure 7 As shown in Figure a, the drug loading capacity of platelets increases in a concentration-dependent manner with increasing Cip concentration, indicating that high concentrations of Cip can enable platelets to load more drugs.

[0089] Furthermore, the drug release pattern of Cip-PLT in the presence and absence of thrombin was investigated. Cip-PLT was incubated in environments with and without thrombin (wt thrombin), and samples were taken at different time points (0–12 h) to determine the proportion of Cip released into the solution. Figure 7 As shown in b, the Cip release rate was faster and the final release amount was higher in the group with thrombin; the Cip release was slower in the group without thrombin. This indicates that thrombin can accelerate the release of Cip from drug-loaded platelets. Thrombin is often present at sites of infection or inflammation, therefore this property facilitates efficient drug release at the target site.

[0090] 2. The efficacy of Cip-PLT in treating bacterial pneumonia refer to Figure 5 The experimental procedure for group a: A bacterial pneumonia model was established in mice by intratracheal inoculation with bacteria; two hours after modeling, different treatments were administered intravenously: the control group received PBS (blank buffer), while the experimental groups received Cip-PLT, PLT, and Cip, respectively; long-term monitoring was then conducted, and the survival rate of each group of mice was recorded. Figure 7 As shown in Figure c, all mice in the PBS and PLT groups died on days 3 and 4, respectively, indicating that unloaded platelets could not treat bacterial pneumonia. Only 10% of mice in the Cip group survived on day 4, indicating that Cip treatment for bacterial pneumonia had some effect, but the effect was weak, possibly due to the poor targeting of antibiotics alone. 80% of mice in the Cip-PLT group still survived on day 5, significantly prolonging the survival time of mice with bacterial pneumonia, indicating that Cip-PLT can more efficiently deliver ciprofloxacin to the site of infection and exert excellent therapeutic effects.

[0091] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A platelet-antibacterial complex, characterized in that, It includes platelets and antimicrobial substances, wherein the platelets load the antimicrobial substances through endocytosis or are coupled with antimicrobial substances through a coupling agent.

2. The platelet antibacterial complex as described in claim 1, characterized in that, The antibacterial substances include bacteriophages and antibiotics.

3. The platelet antibacterial complex as described in claim 2, characterized in that, When the platelets load antimicrobial substances via endocytosis, the antimicrobial substances are antibiotics; when the platelets conjugate antimicrobial substances via a conjugating agent, the antimicrobial substances are bacteriophages.

4. The platelet antibacterial complex as described in claim 3, characterized in that, The coupling agent is tannic acid.

5. The platelet antibacterial complex as described in claim 3, characterized in that, The bacteriophages include one or more of the following bacteriophages that can infect Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae, and Acinetobacter baumannii.

6. The platelet antibacterial complex as described in claim 3, characterized in that, The antibiotics include one or more of the following: penicillins, cephalosporins, macrolides, aminoglycosides, tetracyclines, quinolones, sulfonamides, and glycopeptides.

7. The method for preparing the platelet antibacterial complex according to any one of claims 1-6, characterized in that, Includes the following steps: (1) Platelets are extracted from the blood and prostaglandin E1 is added to the platelets; (2) When the antibacterial substance is bacteriophage, bacteriophage is extracted from the strain, and bacteriophage is mixed with tannic acid to obtain bacteriophage nanoparticles; bacteriophage nanoparticles are incubated with platelets from step (1) to obtain bacteriophage-coupled platelets; (3) When the antibacterial substance is an antibiotic, the antibiotic is incubated with the platelets from step (1) to obtain platelets loaded with antibiotics.

8. The preparation method according to claim 7, characterized in that, The concentration of prostaglandin E1 in step (1) is 1-10 mM; the concentration of bacteriophage in step (2) is 10 mM. 8 -10 11 The concentration of PFU / mL, the concentration of tannic acid is 0.1-20 mg / mL, the particle size of the phage nanoparticles is 50-500 nm; the concentration of the antibiotic in step (3) is 10-1000 μg / mL.

9. The use of the platelet antibacterial complex as described in any one of claims 1-6 in the preparation of a medicament for treating bacterial pneumonia.

10. Use of a composition for the preparation of phage-conjugated platelets, characterized in that, The composition is prostaglandin E1 and tannic acid.

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