A dual-targeting broad-spectrum chimeric bacteriophage antibacterial drug preparation and a preparation method and application thereof

Abstract

The present application belongs to the technical field of biological medicine, and particularly relates to a double-targeted broad-spectrum chimeric bacteriophage antibacterial drug preparation as well as a preparation method and application thereof. The antibacterial drug preparation comprises a biological active substance and a liposome for coating the biological active substance; the biological active substance is a targeted chimeric M13 bacteriophage coupled with an antibacterial substance, and the surface of the liposome is coupled with a targeting antibody. The present application adopts double-targeting design of liposomes and bacteriophages, can specifically target bacterial foci, and loads antibacterial substances on mild M13 bacteriophages, thereby reducing the dosage of antibacterial substances, avoiding uncontrollable release of endotoxins, and finally killing intracellular and extracellular bacteria of foci in the body under the cooperation of liposomes with targeting capability and customizable chimeric bacteriophages.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a dual-targeting broad-spectrum chimeric phage antibacterial drug formulation, its preparation method, and its application. Background Technology

[0002] Antibiotic resistance has become one of the most serious challenges facing modern medicine, a trend that threatens to undermine decades of progress in infection control and clinical treatment. Gram-negative bacteria—such as *Pseudomonas aeruginosa*, *Klebsiella pneumoniae*, and *Acinetobacter baumannii*—are considered the highest priority pathogens due to their significant resistance to almost all classes of antibiotics. These multidrug-resistant pathogens often cause serious diseases such as peritonitis, ventilator-associated pneumonia, and sepsis. The overuse and misuse of broad-spectrum antibiotics, coupled with the near-stagnation of research and development of novel small-molecule antibiotics, further exacerbates this crisis.

[0003] Phage therapy has re-emerged as a promising alternative to traditional antibiotics. Phages are naturally occurring bacterial viruses that selectively infect and lyse host bacteria without harming symbiotic microorganisms, making them inherently precise and self-limiting therapeutic agents. However, the clinical application of phages faces several obstacles: free phages are easily inactivated by environmental factors; their molecular size and charge characteristics result in poor permeability to biofilms and tissues, making it difficult to reach intracellular bacteria and thus hindering targeted killing. Furthermore, current technologies employing lytic phages may exacerbate inflammatory responses due to uncontrolled bacterial lysis and the release of large amounts of endotoxins, a risk particularly pronounced in Gram-negative bacterial infections.

[0004] Therefore, it is necessary to provide an improved phage drug formulation to solve the above problems. Summary of the Invention

[0005] The purpose of this invention is to provide a dual-targeting broad-spectrum chimeric phage antibacterial drug formulation, its preparation method and application. It adopts a dual-targeting design of liposomes and phages, which can specifically target bacterial foci and significantly improve the killing ability of bacteria inside and outside the cell.

[0006] To achieve the above objectives, the first aspect of the present invention provides a dual-targeting broad-spectrum chimeric phage antibacterial drug formulation, comprising: a bioactive substance and liposomes for encapsulating the bioactive substance; wherein the bioactive substance is a targeted chimeric M13 phage coupled with an antibacterial substance, and the liposomes are coupled with a targeting antibody on their surface.

[0007] Furthermore, the targeted chimeric M13 phage is used to target antibodies or peptides targeting pathogens, wherein the pathogens are Gram-negative or Gram-positive bacteria. The targeting antibody conjugated to the surface of the liposome is also an antibody that targets the pathogen.

[0008] Furthermore, the gene sequence of the targeting antibody or targeting peptide has a homologous arm CAGCCGGCCATGGCC at the 5' end and a homologous arm GAACAAAAACTCATC at the 3' end. The targeted chimeric M13 phage protein 3 (pIII protein) expresses the targeted antibody or targeted peptide; The antimicrobial substance is coupled to protein VIII (pⅧ protein) of the targeted chimeric M13 phage.

[0009] Furthermore, the liposomes comprise, by mass percentage, 38%-42% N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride, 33%-37% 1,2-dioleoyl-sn-glycerol-3-phosphate choline, 18%-22% cholesterol, and 3%-7% 1,2-distearate-sn-glycerol-3-phosphate ethanolamine-nitrogen-[succinimide (polyethylene glycol)].

[0010] A second aspect of this invention provides a method for preparing the above-described dual-target broad-spectrum chimeric phage antibacterial drug formulation, comprising the following steps: S1. Homologous recombination of the targeting antibody or targeting peptide with the linearized pADL-10b vector is performed to obtain the modified phage vector plasmid. S2. The phage vector plasmid is transformed into Escherichia coli TG1 competent strain for proliferation culture, then helper phage is added for infection, followed by the addition of kanamycin and inducer, cultured for a preset time, and finally isolated and purified to obtain targeted chimeric M13 phage. S3. Activate the carboxyl groups on the surface of the targeted chimeric M13 phage, and then mix and incubate it with an antibacterial substance to obtain a targeted chimeric M13 phage coupled with an antibacterial substance. S4. The targeted chimeric M13 phage conjugated with antibacterial substances is coated with liposomes, and then a targeting antibody is conjugated on the surface of the liposomes to obtain a dual-targeting broad-spectrum chimeric phage antibacterial drug formulation.

[0011] Furthermore, in step S1, the gene sequence of the targeting antibody or targeting peptide has a homologous arm CAGCCGGCCATGGCC at its 5' end and a homologous arm GAACAAAAACTCATC at its 3' end; the gene sequence of the targeting antibody or targeting peptide is inserted after the PelB signal peptide sequence of the pADL-10b vector.

[0012] Further, step S2 specifically includes: introducing the phage vector plasmid into Escherichia coli TG1 competent strain via electroporation; inoculating the transformed bacterial culture into LB medium containing ampicillin; culturing until the OD600 reaches 0.4-0.6; adding helper phages to the medium and incubating for 1-2 hours to allow the helper phages to infect the bacteria; then adding kanamycin and an inducer to the medium and continuing culturing for 8-16 hours to induce the assembly and secretion of chimeric phages; after culturing, centrifuging, concentrating, and purifying to obtain targeted chimeric M13 phages; The helper phage is CM13D3 or M13KO7 helper phage, and the amount of helper phage added is 0.00005-0.0002 of the LB medium volume; The concentration of kanamycin in the culture medium is 40-55 µg / mL, and the concentration of the inducer is 180-220 µM.

[0013] Furthermore, in step S3, the activation of the carboxyl group specifically includes: blocking the primary amines exposed on the surface of the targeted chimeric M13 phage, and then activating the carboxyl groups on protein 8 of the targeted chimeric M13 phage using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysulfosuccinimide.

[0014] Further, step S4 specifically includes: dissolving 38%-42% of N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride, 33%-37% of 1,2-dioleoyl-sn-glycerol-3-phosphocholine, 18%-22% of cholesterol, and 3%-7% of 1,2-distearyl-sn-glycerol-3-phosphoethanolamine-nitrogen-[succinimide (polyethylene glycol)] in an organic solvent until homogeneous, and then evaporating... The organic solvent is removed, and a liposome film is formed at the bottom. The targeted chimeric M13 phage coupled with antibacterial substances is diluted with PBS buffer and added to the liposome film. After hydration for 20-40 minutes, the liposome film is completely detached by vortexing or blowing to form a milky white multilayer liposome suspension. The suspension is passed through a polycarbonate membrane with a pore size of 200 nm and extruded several times using a liposome extruder to obtain a monolayer liposome suspension. The carboxyl groups on the surface of liposomes were activated using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysulfosuccinimide. Then, a targeting antibody was added, and the mixture was incubated at 3-5°C for 1-2 hours to allow the targeting antibody to covalently bind to the activated liposome surface via amide bonds. The resulting purified product was a dual-targeting broad-spectrum chimeric phage antibacterial drug formulation.

[0015] The third aspect of this invention provides an application of the above-described dual-targeting broad-spectrum chimeric phage antibacterial drug formulation, or the dual-targeting broad-spectrum chimeric phage antibacterial drug formulation obtained by any of the above preparation methods, in the preparation of antibacterial drugs.

[0016] In summary, compared with the prior art, the above-described technical solutions conceived by this invention mainly possess the following technical advantages: 1. The dual-targeting broad-spectrum chimeric phage antibacterial drug formulation provided by this invention utilizes targeted liposomes to encapsulate customizable chimeric phage drugs. This allows for targeting of bacterial infection lesions while also killing drug-resistant bacteria hidden intracellularly. The dual-targeting design of liposomes and phages specifically targets bacterial foci, and by loading antibacterial substances onto mild phages, the amount of antibacterial substance used is reduced, and the uncontrolled release of endotoxins is avoided. Ultimately, the combination of targeted liposomes and customizable chimeric phages can completely eradicate intracellular and extracellular bacteria at foci within the body.

[0017] 2. This invention significantly improves the bactericidal effect on intracellular bacteria by encapsulating liposomes and utilizing their delivery capabilities. Furthermore, by controlling the types and ratios of liposome raw materials and combining this with an extrusion method, the encapsulation effect on larger M13 bacteriophage particles is enhanced.

[0018] 3. Experiments have shown that the M13 chimeric phage dual-targeted antibacterial biological agent prepared in this invention can significantly reduce the total bacterial count and intracellular bacterial count in mice with bacterial peritonitis by killing Pseudomonas aeruginosa. Attached Figure Description

[0019] Figure 1 High-resolution images of phages encapsulated in liposomes; Figure 2 This is a diagram showing the fluorescence colocalization results of chimeric bacteriophages against planktonic Pseudomonas aeruginosa. Figure 3 This is a diagram showing the fluorescence colocalization results of chimeric bacteriophages on Pseudomonas aeruginosa biofilms. Figure 4 The image shows the bactericidal effect of chimeric bacteriophages on Pseudomonas aeruginosa. Figure 5 This is a graph showing the evaluation results of biomembrane penetration efficiency; Figure 6 A diagram showing the results of dual-targeted antibacterial nanoparticles entering cells; Figure 7 The image shows the lysosomal escape results of the dual-targeted antibacterial nanoparticle formulation. Figure 8 This is a diagram illustrating the intracellular bactericidal effect of a dual-targeted antibacterial nanoparticle formulation. Figure 9 This image shows the therapeutic effect of a dual-targeted antibacterial nanoparticle formulation on peritonitis in mice. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0021] The main objective of this invention is to construct a chimeric M13 phage that targets either Gram-negative or Gram-positive bacteria, and to chemically conjugate an antibiotic or other therapeutic agent to protein 8 of this chimeric M13 phage. Simultaneously, the chimeric M13 phage is encapsulated within nanoparticles. The nanoparticle system is not limited to the liposomes described in the examples, but may also include polymer nanoparticles, inorganic nanoparticles, dendritic polymers, etc. Chemically conjugating an antibody to the outside of the liposomes not only overcomes the damage to the phage caused by adverse external environments but also extends the bacterial-killing effect into the cell, improving its therapeutic efficacy against Gram-negative or Gram-positive drug-resistant bacteria.

[0022] The first aspect of this invention provides a universal method for preparing chimeric M13 phages expressing a targeting antibody on protein 3. This method can confer the ability to target a target antigen onto wild-type M13 phages, specifically including the following steps: Step 1: Add a homologous arm CAGCCGGCCAT GGCC to the 5' end of the target protein or polypeptide gene sequence and a homologous arm GAACAAAAACTCATC to the 3' end to synthesize the gene. The synthesized gene fragment is then homologously recombinated with the linearized pADL-10B vector to obtain the modified phage vector plasmid.

[0023] Step 2: The modified phage vector plasmid was electroporated into competent E. coli TG1 strain. During amplification, when the OD600 reached 0.5, helper phage was added at 1 / 1000 of the culture medium volume, and the culture was incubated at 25°C for 1 hour. Then, kanamycin at a final concentration of 50 µg / mL and IPTG inducer at 200 µM were added, and the culture was shaken for 8 hours. Subsequently, targeted chimeric M13 phages were obtained by PEG precipitation or centrifugation concentration.

[0024] The target sequence is selected based on the research object or practical application requirements, and can be an antibody or a short peptide. In some embodiments of the present invention, in order to target the lipopolysaccharide of Gram-negative bacteria, the selected target sequence is the Fab region of Ab-GNB (a targeting antibody fragment targeting Gram-negative bacteria (GNB)). Further, the target fragment is inserted into the pADL-10b vector after the PelB leader peptide, that is, between the CAGCCGGCCATGGCC and ACTAGTGGCCCGGGA sequences.

[0025] Furthermore, antibacterial substances (such as antibiotics) can be coupled to the surface of targeted chimeric M13 phages to enhance antibacterial activity.

[0026] Specifically, this invention utilizes liposomes to encapsulate the aforementioned targeted chimeric M13 phage, overcoming the damage caused by adverse external environments. Simultaneously, the delivery function of the liposomes facilitates intracellular penetration, enhancing the intracellular bacterial killing effect. The specific steps include: DOTAP, DOPC, cholesterol, and DSPE-PEG2000-NHS were dissolved in a mixed solvent of chloroform and methanol, dried under argon to form a thin film at the bottom of the container, and then vacuum dried for 2 hours. Next, the chimeric M13 phage was diluted to the target titer with PBS and added to the liposome film. The mixture was then pipetted to form a milky white suspension, which was subsequently homogenized using a liposome extruder and placed at 4°C to stand.

[0027] In some specific embodiments, the liposome components include 40% DOTAP, 35% DOPC, 20% cholesterol, and 5% DSPE-PEG2000-NHS. Chimeric M13 phages are relatively large, making encapsulation difficult. This invention improves the encapsulation effect by controlling the raw materials and their proportions in the liposomes.

[0028] The chloroform and methanol mixed solvent has a volume ratio of 9:1.

[0029] Using a liposome extruder, you can select PC (polycarbonate) membranes with different pore sizes according to the experimental purpose.

[0030] Furthermore, antibodies are conjugated to the surface of liposomes to prevent the phages from being unable to effectively target the target pathogen after being coated. This includes the following steps: treating the carboxyl groups on the surface of the liposomes with EDC / NHS for 30 minutes to fully activate the groups, adding the targeting antibody and co-incubating for 30 minutes, and then using ultrafiltration centrifugation to remove unbound antibodies.

[0031] Furthermore, the pH of the liposomes needs to be adjusted to between 7.0 and 9.0.

[0032] Furthermore, the molar ratio of EDC:NHS:COOH is 10:10:1.

[0033] The present invention will now be described in detail with reference to the accompanying drawings and embodiments. It should be noted that the scope of protection of the present invention is not limited to the following embodiments, and the reagents used therein, unless otherwise specified, can be obtained through conventional commercial channels.

[0034] 1.1 Experimental Equipment: MicroPulser Electroroporator; PCR instrument (Yisheng NA-2 dual-groove gradient gene amplification instrument); Thermostatic oscillator (Shanghai Minquan Company); Biochemical incubator (Wuhan Ruihua Company); Vacuum drying oven (Wuhan Ruihua Company); Liposome extruder (Mini-Extruder (only), AvantiResearch™-ACrodaBrand); Ultrafiltration centrifuge (Eppendorf); NanoDrop ultraviolet spectrophotometer; Microplate reader (FlexStation3); HITACHI Transmission Electron Microscope; High-resolution microscopy (HIS-SIM); Small animal anesthesia machine (TAIJI); Animal live imaging instrument (AniView600PhoenixX); Confocal microscope (MicroscopeFV3000); Malvern particle size analyzer (Nano-ZS90).

[0035] 1.2 Experimental Materials and Reagents Escherichia coli TG1 electrocompetent cells; CM13D3 helper phage; antibiotics; pADL-10b vector plasmid; DOTAP: N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride, N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride; DOPC: 1,2-Dioleoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine; Ch: Cholesterol; DSPE-PEG2000-NHS: 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[succinimidyl(polyethyleneglycol)-2000], 1,2-disteaaroyl-sn-glycero-3-phosphoethanolamine-N-[succinimidyl(polyethyleneglycol)-2000]; Pseudomonas aeruginosa ATCC15442; BALB / c mice; EDC: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide; Sulfo-NHS: N-Hydroxysulfosuccinimide, N-hydroxysulfosuccinimide.

[0036] Example 1: Preparation of targeted chimeric M13 phage loaded with antibacterial substances.

[0037] Step 1: Gene Fragment Synthesis and Vector Construction. Based on the target pathogen (Gram-negative or Gram-positive bacteria), select or screen suitable targeting antibodies or peptides. Antibody sequences can be obtained by searching publicly available databases; alternatively, phage display technology can be used to screen for desired targeting peptides, and the gene sequence of the targeting peptide fragment can be obtained through sequencing (in this example, the Fab region of Ab-GNB is used). The gene sequence is then augmented with designated homologous arms (CAGCCGGCCATGGCC and GAACAAAAACTCATC) at the 5' and 3' ends, respectively. The PADL-10B vector is linearized, and the synthesized gene fragment is inserted into the linearized vector after the PelB signal peptide sequence (i.e., between CAGCCGGCCATGGCC and ACTAGTGGCCCGGGA) using homologous recombinase to obtain the recombinant phage plasmid. The correctness of the recombinant plasmid sequence is verified by first-generation sequencing.

[0038] Step 2: Assembly and amplification of chimeric phages. The validated recombinant phage plasmid was introduced into *E. coli* TG1 competent cells via electroporation. The transformed bacterial culture was inoculated into LB medium containing ampicillin and cultured at 37°C with shaking until the OD600 (optical density at 600 nm) reached approximately 0.5. Then, 0.1% (relative to the total volume of the medium) of CM13D3 helper phage was added to the culture, gently mixed, and incubated at 25°C for 1 hour to allow the helper phage to infect the bacteria. Kanamycin and an inducer (IPTG) were added to the medium to achieve a kanamycin concentration of 50 µg / mL and an IPTG concentration of 200 µM. The culture was continued at 25°C with shaking at 250 rpm for 8–16 hours to induce the assembly and secretion of chimeric phages. After the culture was completed, the supernatant was collected by centrifugation. The chimeric M13 phage in the supernatant was concentrated and purified by PEG / NaCl precipitation or ultracentrifugation. The phage precipitate was resuspended in PBS, its titer (PFU / mL) was determined, and it was aliquoted and stored at -80℃.

[0039] Step 3: Chemical coupling of antimicrobial substances to the phage surface. To prevent the primary amine on the anti-LPS antibody from reacting with the subsequent cross-linking reagent and causing phage cross-linking and aggregation, the chimeric phage obtained in Step 2 was first treated with sulfo-NHS-acetate. The reaction system was incubated at room temperature in the dark for 30 minutes to block the primary amine exposed on the antibody surface. Excess sulfo-NHS-acetate was then removed by dialysis. The reaction product was purified by dialysis using a Slide-A-LyzerMini dialysis apparatus (20 KMWCO) at 4°C. Dialysis was performed twice in 1xPBS buffer (pH 7.2) and then three times in 0.1M MES buffer (pH 5.5) to provide the optimal pH environment for subsequent EDC / Sulfo-NHS activation. Each dialysis cycle lasted 4 hours.

[0040] Activation of carboxyl groups on the phage surface: Transfer the dialyzed phage solution to a new container. Add a freshly prepared aqueous solution of EDC (final concentration approximately 4 mg / mL) and Sulfo-NHS (final concentration approximately 11 mg / mL), and incubate with gentle stirring at room temperature for 20 minutes. This step aims to activate the carboxyl groups on the phage capsid proteins (especially pVIII protein). To remove unreacted EDC and Sulfo-NHS, rapidly dialyze the activated phage solution three times at 4°C with 1×PBS buffer (pH 7.2), 40 minutes each time.

[0041] Antimicrobial conjugation: In this example, polymyxin B (PMB) is used as the antimicrobial agent. The dialysis product is transferred to a 15 mL centrifuge tube, and the volume is brought up to 10 mL with 1xPBS buffer (pH 7.2). Subsequently, the conjugation is performed at 1 mL per phage solution (10... 12 Viral particles were added at a ratio of 20 mg PMB, along with polymyxin B. The mixture was gently mixed at room temperature and incubated for 2 hours to allow the primary amine of PMB to form a stable amide bond with the carboxyl groups on the activated phage surface. Reaction termination and purification: After the coupling reaction was complete, the sample was dialyzed twice with 1xTBS buffer (pH 7.2) to quench any residual reactivity. Finally, it was dialyzed three times with 1xPBS buffer (pH 7.4) to completely remove unreacted, free polymyxin B molecules, yielding purified PMB-chimeric M13 phage. Coupling efficiency verification: The purified PMB-chimeric M13 phage sample was subjected to mass spectrometry analysis by a third-party institution to quantitatively confirm the successful coupling of PMB.

[0042] Example 2: Preparation of nanoparticles loaded with antibacterial substances-chimeric phages.

[0043] Step 1: Formation of the lipid film. In this example, liposomes are used as nanoparticles. Each component is prepared to a stock solution concentration of 100 mM. The lipid components are precisely pipetted by weight percentage (40% DOTAP, 35% DOPC, 20% cholesterol, 5% DSPE-PEG2000-NHS) and placed in a round-bottom flask. A chloroform:methanol (9:1, v / v) mixture is added to fully dissolve the components. Using a rotary evaporator at a suitable water bath temperature (e.g., 40°C), argon or nitrogen gas is introduced to slowly evaporate and remove the organic solvent until a uniform, transparent lipid film forms on the flask wall. The flask is then placed in a vacuum desiccator overnight (at least 2 hours) to completely remove any residual organic solvent.

[0044] Step 2, hydration and extrusion. The chimeric phage prepared in Example 1 was diluted with PBS buffer to the target titer (e.g., 1 × 10⁻⁶). 11 (PFU / mL). This phage suspension was added to a flask containing a lipid film and allowed to hydrate for 30 minutes at room temperature or in a water bath above the lipid phase transition temperature. Subsequently, the lipid film was completely detached by gentle vortexing or pipetting, forming a milky white multilayer liposome suspension. To ensure uniform liposome particle size, this suspension was passed through a 200 nm polycarbonate membrane and extruded multiple times using a liposome extruder. The resulting uniform small monolayer liposome (SUV) suspension was stored at 4°C for later use.

[0045] Example 3: Antibody modification on the surface of liposomes.

[0046] Step 1, Activation. Take an appropriate amount of the liposome suspension prepared in Example 2, adjust the concentration with PBS or MES buffer at pH 7.4-8.0, and add freshly prepared EDC and NHS aqueous solution according to the molar ratio of EDC:NHS:mPEG-COOH on the liposomes of 10:10:1. Stir gently at room temperature for 30 minutes to activate the carboxyl groups on the surface of the liposomes.

[0047] Step 2, Coupling and Purification. An excess of the targeting antibody was directly added to the activated liposome reaction solution. In this example, the antibody targeted LPS, and the addition ratio was 5 times the NHS groups on the liposome surface. Incubation was continued at 4°C for 1-2 hours to allow the antibody to covalently bind to the activated liposome surface via amide bonds. After the reaction, unreacted antibody, EDC, NHS, and byproducts were removed using ultrafiltration centrifuge tubes (e.g., with a 60 kDa molecular weight cutoff) or gel size exclusion chromatography, yielding a purified, antibody-modified, internally encapsulated chimeric phage dual-targeting antibacterial biopharmaceutical. The final product was characterized by particle size, zeta potential, and phage encapsulation efficiency.

[0048] Example 4: Characterization and Evaluation of Dual-Targeted Antibacterial Nanoparticles Phage-labeled fluorescence: 1 mL of 10 12 The PMB-chimeric M13 phage prepared in Example 1 (CFU / ml) and 2 μL of PE (phycoerythrin) fluorescently labeled M13 phage antibody were incubated in 1 mL PBS buffer (pH 7.0) with gentle stirring overnight at room temperature. The free dye was removed by extensive dialysis (MWCO 3500 Da) in 500 mL PBS buffer (pH 7.0), and the bioconjugate was ultrafiltered to approximately 200 µL using an Amicon Ultra-4 10000 filter to obtain fluorescently labeled phage M13-PE.

[0049] Liposome-labeled fluorescence: Each component was prepared to a stock solution concentration of 100 mM. Each lipid component was precisely pipetted by weight percentage (40% DOTAP, 35% DOPC, 20% cholesterol, 5% DSPE-PEG2000-FITC (fluorescein isothiocyanate)) and placed in a round-bottom flask. A chloroform:methanol mixture (9:1, v / v) was added to fully dissolve the components. Using a rotary evaporator at a suitable water bath temperature (e.g., 40°C), argon or nitrogen gas was introduced to slowly evaporate and remove the organic solvent until a uniform, transparent lipid film formed on the flask wall. The flask was placed in a vacuum desiccator overnight (at least 2 hours) to completely remove any residual organic solvent. Then, the liposome-FITC film was resuspended in the fluorescently labeled bacteriophage (M13-PE). The film was gently vortexed or blown off to completely detach the lipid film, forming a milky white, multilayered liposome suspension. To ensure uniform liposome particle size, the suspension was passed through a 200 nm polycarbonate membrane and extruded multiple times using a liposome extruder to obtain a uniform small monolayer liposome (SUV) suspension, which was then imaged using a super-resolution microscope (SIM).

[0050] The results are as follows Figure 1 As shown, liposomes (green fluorescence, Figure 1 The liposome-FITC successfully encapsulated the bacteriophage (red fluorescence) inside.

[0051] Example 5: Validation of the targeting effect on planktonic bacteria.

[0052] Phage-labeled fluorescence: 1 mL of 10 12 CFU / mL of PMB-chimeric phage was incubated with 2 μL of PE fluorescently labeled M13 phage antibody (Anti-M13 Antibody (PE)) in 1 mL of PBS buffer (pH 7.0) and gently stirred overnight at room temperature. Free dye was removed by extensive dialysis (MWCO 3500 Da) in 500 mL of PBS buffer (pH 7.0), and the bioconjugate was ultrafiltered to approximately 200 µL using an Amicon Ultra-4 10000 filter to obtain PE-LPSphages.

[0053] Fluorescent labeling of planktonic bacteria: In this example, *Pseudomonas aeruginosa* ATCC15442 was used. 1 mL of *P. aeruginosa* ATCC15442 with an OD600 of 0.6 was co-incubated with 2 μL of rabbit polyclonal anti-pseudomonas aeruginosa (Abcamab68538) diluted 1:1000 for 1.5 h, washed three times with PBS, and then incubated with the secondary antibody goat anti-rabbit IgG H&L (Alexa Fluor® 488) (ab150077) for 1.5 h, washed three times with PBS. The labeled bacteria and bacteriophages were co-incubated for 30 minutes, with a total volume of 1 mL, and co-localization was observed under a confocal microscope.

[0054] The results are as follows Figure 2 As shown, free bacteriophages, labeled with PE fluorescent dye, appear red, while Pseudomonas aeruginosa, labeled with Alexa Fluor® 488 dye, appears green. Merging the two channels reveals fluorescent co-localization between the chimeric bacteriophage and the planar Pseudomonas aeruginosa, indicating that the bacteriophage successfully targeted the planar bacteria.

[0055] Example 6: Validation of the targeting effect on bacterial biofilms.

[0056] Phage-labeled fluorescence: 1 mL of 10 12 CFU / ml of PMB-chimeric phages and 2 μL of PE fluorescently labeled M13 phage antibody (Anti-M13 Antibody (PE)) were incubated in 1 mL of PBS buffer (pH 7.0) with gentle stirring overnight at room temperature. Free dye was removed by extensive dialysis (MWCO 3500 Da) in 500 mL of PBS buffer (pH 7.0), and the bioconjugate was ultrafiltered to approximately 200 µL using an Amicon Ultra-4 10000 filter to obtain PE-LPSphages.

[0057] Fluorescent labeling of bacterial biofilms: This example uses *Pseudomonas aeruginosa*. When the bacteria reach an OD600 of 0.6, 150 μl is added to a confocal microplate and incubated overnight at 37°C. The culture medium is then discarded, and the biofilm is washed twice with double-distilled water. 500 ml (1:1000 dilution) of rabbit polyclonal anti-Pseudomonas aeruginosa antibody (ab68535) is added to the biofilm and incubated for 1.5 hours. After incubation, the antibody is aspirated and washed three times with PBS. The biofilm is then incubated with goat anti-rabbit IgG H&L-488 (ab1500773) for 1.5 hours, aspirated, and washed three times with PBS. The fluorescently labeled phages are co-incubated with the biofilm for 30 minutes, observed under FV3000, and 3D images are captured.

[0058] The results are as follows Figure 3As shown, free bacteriophages, labeled with PE fluorescent dye, appear red, while biofilms formed by Pseudomonas aeruginosa, labeled with Alexa Fluor® 488 dye, appear green. By scanning the biofilm layer by layer and then using the software's built-in function to generate a stacked image, the overlap of the two channels reveals a fluorescent co-localization phenomenon between the Pseudomonas aeruginosa biofilm and the chimeric bacteriophages. The results indicate that the bacteriophages successfully targeted the bacterial biofilm.

[0059] Example 7: Verification of Bactericidal Effect Step 1: Inoculate a single colony of *Pseudomonas aeruginosa* into LB medium and amplify it at 37°C and 200 rpm. Remove the colony when the OD600 reaches 0.5, convert the concentration to bacterial concentration using the formula, and dilute with PBS to a concentration of 10⁻⁶. 6 CFU / mL.

[0060] Step two: The treatment experiment was set up into three groups as follows: no treatment, PMB treatment, and M13-PMB (PMB-modified targeted chimeric M13 phage) treatment. The antibiotic concentration should be slightly higher than the minimum inhibitory concentration (MIC) of the bacteria. In this example, for Pseudomonas aeruginosa, the PMB concentration was set at 0.05 ug / mL. The bacteria and experimental groups were incubated for different time gradients: 0, 0.5, 1, 2, 3, 6, 9, and 12 hours. Then, the mixture was diluted 1:1000, and 100 μL was plated on LB agar plates and incubated at 37°C for 16 hours before photographing and counting.

[0061] The results are as follows Figure 4 As shown, in the group of Pseudomonas aeruginosa without any treatment (AFI), the bacterial count was clearly visible at all time points; in the group treated with PMB (AFI+PMB), the bacterial count showed a decreasing trend in the first three hours, but began to increase again as time went on; in the group with PMB-modified targeted phages (AFI+M13-PMB), no bacterial growth was found in the smears from 0.5 hours to 12 hours, indicating a significant bactericidal effect, which was better than using antibiotics alone and could reduce the concentration and dosage of antibiotics used.

[0062] Example 8: Evaluation of biomembrane penetration efficiency Step 1: In this embodiment, Pseudomonas aeruginosa is used as an example of Gram-negative bacteria. Fresh bacterial solution with an OD600 of 0.5 is added to a confocal dish and incubated at 37°C for 48 hours to form a biofilm. Plankton is removed by washing three times with PBS.

[0063] Step two: Phages were stained with Anti-M13 Antibody (PE), and the Pseudomonas aeruginosa constructs exhibited GFP fluorescence. The biofilm therapeutic experiments were set up in three groups: M13 phage, LCP (liposome-encapsulated chimeric phage loaded with antibacterial substances and targeting lipopolysaccharide), and Ab-LMP (liposome-encapsulated chimeric phage loaded with antibacterial substances and targeting lipopolysaccharide modified with a targeting antibody). The biofilms and therapeutic materials were co-incubated for 3 hours. Penetration profile studies were performed using an Olympus confocal scanning microscope, and the images were processed using ImageJ.

[0064] The results are as follows Figure 5 As shown, Ab-LMP penetrates the biofilm to the deepest depth, superior to LCP. This is because the liposomes have targeting antibodies on their surface, which are more specific to bacteria and can more easily reach the depths of the biofilm.

[0065] Example 9: Dual-targeted antibacterial nanoparticles can enter cells and escape via lysosomes.

[0066] Cell entry: Seed 50,000 HEK293 cells per well into confocal microscopy dishes and culture overnight until adherent. Label the phages with PE fluorescence as described in Example 5. The experiment was divided into two groups: M13 and LCP (liposome-encapsulated chimeric phages loaded with antibacterial substances and targeting lipopolysaccharide). Discard the culture medium in the confocal microscopy dishes and replace it with a final concentration containing 10... 9 M13 phage at CFU / mL was co-incubated with LCP culture medium for 1, 2, and 4 hours. After each incubation period, the cells were washed three times with PBS. Then, the cell nuclei were stained with Hoechst dye (100×) for 10 minutes. The supernatant was removed, and the cells were washed three times with PBS. Finally, the cells were immersed in 500 μL of PBS for observation under a confocal microscope.

[0067] The results are as follows Figure 6 As shown, M13 lacks liposome encapsulation and cannot enter the cell, while LCP can successfully carry bacteriophages into the cell, possessing the potential for intracellular bactericidal activity.

[0068] Lysosomal escape: 50,000 HEK293 cells were seeded per well into confocal microscopy dishes and cultured overnight until adherent. Phages were then labeled with PE fluorescence following the steps in Example 4. The culture medium in the confocal microscopy dishes was discarded and replaced with a final concentration of 10... 10The medium was incubated with LCP at CFU / mL for 1-2 hours. After each incubation period, the cells were washed three times with PBS. Then, the nuclei and lysosomes were stained with Hoechst dye (100×) and Lyso Tracker Green (lysosomal green fluorescent probe). The supernatant was removed, and the cells were washed three times with PBS. Finally, the cells were immersed in 500 μL of PBS for observation under a confocal microscope.

[0069] The results are as follows Figure 7 As shown, within one hour, the bacteriophage still exhibited co-localization with the lysosome, indicating that the bacteriophage was still inside the lysosome. After two hours, it could be observed that the bacteriophage and lysosome had not co-localized, indicating that the bacteriophage had been released from the lysosome.

[0070] Example 10: Verification of the intracellular bactericidal effect of dual-targeted antibacterial nano-formulation Step 1: Establishment of a bacterial infection cell model. The MOI (Mean Interval) of *Pseudomonas aeruginosa* is typically 1–100. Cell infection is performed using an MOI of 50, for example, 7.5 × 10⁻⁶ cells. 5 Each cell requires 3.75 × 10⁻⁶ cells. 7 The bacteria were infected for 16 hours. The culture medium was then discarded, and the extracellular bacteria were washed away with PBS. The bacteria were then incubated with the corresponding antibiotic for 2 hours to completely kill the free extracellular bacteria. The culture medium was then washed three times with PBS and replaced with fresh DMEM medium.

[0071] Step 2: Verification of intracellular bactericidal effect. The experiment was set up in the following groups: PBS, M13, LCP, and Ab-LCP, for a total of 4 groups. The bacteriophages were labeled with PE fluorescence according to the steps in Example 4, and the bacteriophage concentration was set to 10. 10 CFU / mL was used for co-incubation treatment. The supernatant was replaced with PBS at 2 hours and 4 hours, followed by confocal microscopy imaging.

[0072] The results are as follows Figure 8 As shown, mCherry red fluorescence expressed by bacteria was observed in the cells of the PBS and M13 groups, indicating that bacteria were still present in the cells. However, no mCherry red fluorescence expressed by bacteria was observed in the cells of the LCP and Ab-LCP groups, indicating that the bacteria in the cells were successfully killed.

[0073] Example 11: Therapeutic effect of dual-targeted antibacterial nano-formulation on mouse peritonitis Step 1: Establishment of a mouse model of peritonitis. Animals used were 6-7 week old male Balb / c mice. In this example, *Pseudomonas aeruginosa* was used as an example, with 5 × 10⁻⁶ bacteria... 6Mice were injected intraperitoneally with a bacterial dose of CFU / mouse and fed normally for 24 hours. The mice were divided into 6 groups: the control group was not injected with bacteria, and the remaining 5 groups were injected with bacteria but treated with different materials, namely PBS, PMB, M13-PMB, LCP, and Ab-LCP.

[0074] Step 2: Mice injected with bacteria were randomly divided into 5 groups (n=5). Each group received a single injection of a different drug treatment for 48 hours, including PBS, PMB, M13-PMB, LCP, and Ab-LCP. Finally, the mice were injected with 2 mL of cold HBSS and then sacrificed. Peritoneal fluid was collected to determine total CFU, extracellular CFU, and intracellular CFU. One-third of the peritoneal fluid was used to quantify total colony-forming units (CFUS); the supernatant of one-third of the peritoneal fluid was collected after centrifugation and used to quantify extracellular CFUS; the remaining one-third of the peritoneal fluid was co-incubated with antibiotics to kill extracellular MRSA, and then lysed with HBSS solution containing 0.1% bovine serum albumin (BSA) and 0.1% Tween X-100 to quantify intracellular CFUS.

[0075] The results are as follows Figure 9 As shown, the total bacterial count was lowest and the effect was best with Ab-LCP. The extracellular bacterial count was significantly lower with Ab-LCP compared to LCP, indicating that the liposome-modified antibody was more likely to find the bacterial lesion site and release the chimeric phage inside the liposome. The intracellular bacterial count was significantly lower in both LCP and Ab-LCP groups than in other groups without liposome encapsulation, indicating that the addition of liposomes provided key support for the killing of intracellular bacteria.

[0076] In summary, this invention utilizes *Pseudomonas aeruginosa* ATCC15442 to establish a Gram-negative bacteria-HEK293 cell infection model, demonstrating that the M13 chimeric phage dual-targeting antibacterial biopharmaceutical exhibits better control over intracellular bacteria compared to free chimeric phages. Specifically, when cells infected with *P. aeruginosa* were simultaneously incubated with phages coated with liposomes and free chimeric phages at the same concentration for 2 hours, the liposome-coated phages reduced the number of *P. aeruginosa* ATCC15442 cells by up to 21.2% compared to naked phages. This invention can improve the killing or inhibitory effect on both Gram-negative planktonic and intracellular bacteria while reducing the amount of antibiotics used.

[0077] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a dual-targeting broad-spectrum chimeric phage antibacterial drug formulation, characterized in that, Includes the following steps: S1. The targeting antibody or targeting peptide is homologously recombinated with the linearized pADL-10b vector to obtain the modified phage vector plasmid; the gene sequence of the targeting antibody or targeting peptide has a homologous arm CAGCCGGCCATGGCC at the 5' end and a homologous arm GAACAAAAACTCATC at the 3' end; the gene sequence of the targeting antibody or targeting peptide is inserted after the PelB signal peptide sequence of the pADL-10b vector. S2. The phage vector plasmid is transformed into Escherichia coli TG1 competent strain for proliferation culture, then helper phage is added for infection, followed by the addition of kanamycin and inducer, cultured for a preset time, and finally isolated and purified to obtain targeted chimeric M13 phage. S3. Activate the carboxyl groups on the surface of the targeted chimeric M13 phage, and then mix and incubate it with an antibacterial substance to obtain a targeted chimeric M13 phage coupled with an antibacterial substance; the antibacterial substance is coupled to protein 8 of the targeted chimeric M13 phage; the antibacterial substance is polymyxin B; S4. Dissolve 38%-42% of N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride, 33%-37% of 1,2-dioleoyl-sn-glycerol-3-phosphocholine, 18%-22% of cholesterol, and 3%-7% of 1,2-distearate-sn-glycerol-3-phosphoethanolamine-nitrogen-[succinimide (polyethylene glycol)] in an organic solvent until homogeneous. Then evaporate the organic solvent to form a liposome film at the bottom. Dilute the targeted chimeric M13 phage coupled with antibacterial substances with PBS buffer and add it to the liposome film. Hydrate for 20-40 min. Then vortex or blow to completely detach the liposome film to form a milky white multilayer liposome suspension. Pass the suspension through a polycarbonate membrane with a pore size of 200 nm and extrude it several times using a liposome extruder to obtain a monolayer liposome suspension. The carboxyl groups on the surface of liposomes were activated using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysulfosuccinimide. Then, an antibody targeting LPS was added, and the mixture was incubated at 3-5°C for 1-2 hours. This allowed the antibody targeting LPS to covalently bind to the activated liposome surface via amide bonds. The resulting purified drug was a dual-targeting broad-spectrum chimeric phage antibacterial agent.

2. The method for preparing the dual-targeting broad-spectrum chimeric phage antibacterial drug formulation according to claim 1, characterized in that, Step S2 specifically includes: introducing the phage vector plasmid into Escherichia coli TG1 competent strain via electroporation; inoculating the transformed bacterial culture into LB medium containing ampicillin; culturing until the OD600 reaches 0.4-0.6; adding helper phages to the medium and incubating for 1-2 hours to allow the helper phages to infect the bacteria; then adding kanamycin and an inducer to the medium and continuing culturing for 8-16 hours to induce the assembly and secretion of chimeric phages; after culturing, centrifuging, concentrating, and purifying to obtain targeted chimeric M13 phages; The helper phage is CM13D3 or M13KO7 helper phage, and the amount of helper phage added is 0.00005-0.0002 of the LB medium volume; The concentration of kanamycin in the culture medium is 40-55 µg / mL, and the concentration of the inducer is 180-220 µM.

3. The method for preparing the dual-targeting broad-spectrum chimeric phage antibacterial drug formulation according to claim 1, characterized in that, In step S3, the activation of the carboxyl group specifically includes: blocking the primary amines exposed on the surface of the targeted chimeric M13 phage, and then activating the carboxyl groups on protein 8 of the targeted chimeric M13 phage using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysulfosuccinimide.

4. A dual-targeting broad-spectrum chimeric phage antibacterial drug formulation, characterized in that, It is obtained by the preparation method according to any one of claims 1-3.

5. The application of a dual-targeting broad-spectrum chimeric phage antibacterial drug formulation obtained by the preparation method according to any one of claims 1-3 in the preparation of antibacterial drugs.

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