Antibacterial protein nano-drug delivery system as well as preparation method and application thereof

Abstract

The invention discloses an antibacterial protein nano-drug delivery system as well as a preparation method and application thereof, and relates to the technical field of biological medicines. The preparation method of the antibacterial protein nano drug delivery system comprises the following steps: carrying out mixed reaction on liposome encapsulating antibacterial protein CB6-C and an erythrocyte membrane to obtain RBCM-CB6-C-NLCs liposome; the RBCM-CB6-C-NLCs liposome and a PBP2a monoclonal antibody are subjected to a mixed reaction, and the antibacterial protein nano drug delivery system is obtained; the amino acid sequence of the antibacterial protein CB6-C is as shown in SEQ ID NO. 1. The drug delivery system provided by the invention can achieve the purposes of slowly releasing drugs, avoiding immune cell phagocytosis and actively targeting MRSA, has good safety and bactericidal activity, and has a good treatment effect on MRSA infection.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an antibacterial protein nano-delivery system, its preparation method, and its application. Background Technology

[0002] Staphylococcus aureus, a significant pathogen, derives its pathogenicity primarily from the synergistic effects of multiple virulence factors, enabling it to cause a wide range of clinical symptoms, from mild infections to fatal diseases. Due to antibiotic overuse and continuous evolution, the emergence and spread of methicillin-resistant Staphylococcus aureus (MRSA) poses a serious challenge to the current healthcare system. This pathogen can cause a variety of illnesses, ranging from moderately severe skin infections to fatal pneumonia and sepsis.

[0003] First-line drugs widely used in clinical practice for MRSA infection mainly include glycopeptides (such as vancomycin and teicoplanin), linezolid, and daptomycin. These drugs have significantly improved the clinical treatment outcomes for patients with MRSA infection. However, with the continuous evolution of MRSA under the pressure of antimicrobial drugs, highly resistant strains have emerged clinically, and adverse drug reactions have occurred during long-term treatment. Therefore, traditional drug therapy is insufficient to address the increasingly severe challenge of MRSA infection, and the search for novel anti-MRSA drugs with strong antimicrobial activity and high safety and stability has become a hot topic in scientific research. In recent years, research has found that antimicrobial proteins have advantages such as rapid killing of pathogenic microorganisms, broad antimicrobial spectrum, and low resistance rate. However, their short half-life and susceptibility to enzymatic degradation have limited their widespread clinical application. With the continuous evolution of MRSA under the pressure of antimicrobial drugs, the problem of drug resistance is becoming increasingly serious. Developing a novel antimicrobial drug with strong anti-MRSA activity, high safety and stability, and the ability to actively target pathogens is urgently needed. Summary of the Invention

[0004] The purpose of this invention is to provide an antimicrobial protein nanoparticle drug delivery system, its preparation method, and its applications, in order to solve the problems existing in the prior art. The drug delivery system provided by this invention can achieve slow drug release, avoid phagocytosis by immune cells, and actively target MRSA, and has good safety and bactericidal activity, showing good therapeutic effects against MRSA infection.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides a method for preparing an antibacterial protein nano-drug delivery system, comprising the following steps:

[0007] The liposomes encapsulating the antimicrobial protein CB6-C were mixed with the erythrocyte membrane to obtain RBCM-CB6-C-NLCs liposomes;

[0008] The RBCM-CB6-C-NLCs liposomes were mixed with PBP2a monoclonal antibody to obtain the antibacterial protein nano-delivery system.

[0009] The amino acid sequence of the antimicrobial protein CB6-C is shown in SEQ ID NO.1.

[0010] Furthermore, the method for preparing the liposomes encapsulating the antimicrobial protein CB6-C includes the following steps:

[0011] The antimicrobial protein CB6-C, lecithin, and cholesterol are dissolved in a solvent. After the solvent is evaporated, water is added for hydration to obtain liposomes encapsulating the antimicrobial protein CB6-C.

[0012] Furthermore, the mass ratio of the antimicrobial protein CB6-C, the lecithin, and the cholesterol is 4:5:1.

[0013] Further, the solvent includes anhydrous ethanol and dichloromethane; and / or

[0014] The volume ratio of the anhydrous ethanol to the dichloromethane is 5:1.

[0015] Furthermore, the mass ratio of the red blood cell membrane to the liposome encapsulating the antimicrobial protein CB6-C is (1-4):10.

[0016] Preferably, the mass ratio of the red blood cell membrane to the liposomes encapsulating the antimicrobial protein CB6-C is 3:10.

[0017] The present invention also provides an antibacterial protein nano-drug delivery system prepared according to the above preparation method.

[0018] The present invention also provides the application of the above-described antimicrobial protein nano-delivery system in the preparation of antimicrobial drugs.

[0019] The present invention also provides an antibacterial drug, including the above-described antibacterial protein nano-delivery system.

[0020] Furthermore, the antimicrobial drug also includes pharmaceutically acceptable excipients.

[0021] The present invention discloses the following technical effects:

[0022] This invention utilizes a strategy of encapsulating antimicrobial proteins in nanoliposomes and coupling them with target molecules. By coupling a penicillin-binding protein 2a (PBP2a) monoclonal antibody that specifically binds to MRSA to the surface of liposomes and to erythrocyte membranes, and then coating the antimicrobial protein CB6-C, an antimicrobial protein nano-delivery system (CB6-C-NLCs-MRM) is constructed. Loading the antimicrobial protein CB6-C into liposomes improves water solubility and enhances the therapeutic effect against MRSA. Coating the erythrocyte membrane onto the surface of the CB6-C liposomes, through protein modification of the erythrocyte membrane surface, avoids macrophage uptake, effectively prolonging the in vivo circulation time of the antimicrobial protein, thereby improving the therapeutic effect against MRSA. The PBP2a monoclonal antibody loaded into this delivery system can target and capture MRSA, further improving the therapeutic effect against MRSA infection.

[0023] The drug delivery system provided by this invention can achieve slow drug release, avoid phagocytosis by immune cells, and actively target MRSA. It also has good safety and bactericidal activity, and has a good therapeutic effect on MRSA infection. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 Statistical graphs showing the encapsulation efficiency (A) and drug loading (B) of liposomes under different drug-liposome ratios;

[0026] Figure 2 Statistical graphs showing the encapsulation efficiency (A) and drug loading (B) of liposomes under different phospholipid ratios;

[0027] Figure 3 Statistical graphs showing the encapsulation efficiency (A) and drug loading (B) of liposomes under different ratios of anhydrous ethanol to dichloromethane;

[0028] Figure 4 Statistical graphs showing the encapsulation efficiency (A) and drug loading (B) of liposomes under different hydration time conditions;

[0029] Figure 5 Statistical graphs of liposome encapsulation efficiency (A) and drug loading rate (B) under different ultrasound time conditions;

[0030] Figure 6Statistical graphs of liposome encapsulation efficiency (A) and drug loading rate (B) under different ultrasound power conditions;

[0031] Figure 7 The images show the morphological observation and identification results of erythrocytes and erythrocyte membranes in Example 3; where A is a regular optical microscope image of erythrocytes (RBCs) (40×10); B is a regular optical microscope image of erythrocytes (RBCs) after Wright staining (40×10); C is the SDS-PAGE analysis results of erythrocytes and erythrocyte membranes, M: protein marker; 1: erythrocyte; 2: erythrocyte membrane;

[0032] Figure 8 Statistical graphs showing the encapsulation efficiency (A) and drug loading efficiency (B) of CB6-C-NLCs-MRM under different red blood cell membrane to liposome mass ratios;

[0033] Figure 9 Figure 1 shows the surface protein characterization results of CB6-C-NLCs-MRM. In Figure 2, A represents the SDS-PAGE analysis results: M: Protein Marker, 1: CB6-C, 2: NLCs, 3: CB6-C-NLCs, 4: RBCM-CB6-C-NLCs, 5: CB6-C-NLCs-MRM, 6: RBCM, 7: RBC. Figure 3 shows the results of Western blotting analysis of Band3 protein expression in RBCM-CB6-C-NLCs, CB6-C-NLCs-MRM, RBCM, and RBC: M: Protein Marker, 1: RBCM-CB6-C-NLCs, 2: CB6-C-NLCs-MRM, 3: Erythrocytes, 4: Erythrocyte membrane.

[0034] Figure 10 Particle size (A) and potential (B) distributions of CB6-C-NLCs;

[0035] Figure 11 Particle size (A) and potential (B) distributions of RBCM-CB6-C-NLCs;

[0036] Figure 12 Particle size (A) and potential (B) distributions of CB6-C-NLCs-MRM;

[0037] Figure 13 Morphological characterization of CB6-C-NLCs-MRM (A) and TEM image (B);

[0038] Figure 14 XRD patterns of each component in CB6-C-NLCs-MRM;

[0039] Figure 15 FTIR scans of each component in CB6-C-NLCs-MRM;

[0040] Figure 16 The in vitro drug release profiles of CB6-C-NLCs, RBCM-CB6-C-NLCs, and CB6-C-NLCs-MRM in PBS buffer (pH=7.4) are shown.

[0041] Figure 17 Growth curve (A) and bactericidal curve (B) of CB6-C-NLCs-MRM against MRSA;

[0042] Figure 18 The graph shows the results of the hemolytic activity assay for CB6-C-NLCs-MRM.

[0043] Figure 19 The graph shows the cytotoxicity assay results of CB6-C-NLCs-MRM.

[0044] Figure 20 The results show the effects of CB6-C-NLCs-MRM injection on mouse body weight, food intake, and water intake; where A is the body weight change curve; B is the statistical graph of food intake; and C is the statistical graph of water intake.

[0045] Figure 21 The images show the histopathological analysis of acute toxicity in mice (A) and the results of H&E staining (B) (×400).

[0046] Figure 22 The graph shows the results of detecting the effects of drug injection on mouse body weight, food intake, and water intake during the subchronic toxicity study; where A is the body weight change curve; B is the statistical graph of food intake; and C is the statistical graph of water intake.

[0047] Figure 23 The histopathological analysis of subchronic toxicity in mice (A) and the results of H&E staining (B) (×400) are shown.

[0048] Figure 24 The image shows the results of RAW264.7 cells uptake of different nanoliposome suspensions; where A is a laser confocal image of cell uptake (scale bar: 25μm; red: DiD; blue: cell nucleus); B is a flow cytometry analysis image.

[0049] Figure 25 The graph shows the results of MRSA uptake of different nanosuspensions; where A is a laser confocal micrograph of cellular uptake (scale bar: 25μm; red: DiD; blue: cell nucleus); and B is a flow cytometry analysis graph.

[0050] Figure 26 In vivo fluorescence imaging images of mice from different groups;

[0051] Figure 27 Fluorescence imaging of organs in mice from different groups;

[0052] Figure 28 The graphs show the fluorescence intensity and bacterial load of mice in different groups; AC represents the fluorescence intensity of the liver, spleen, and kidney at different time points; DF represents the bacterial load at 4h, 24h, and 48h.

[0053] Figure 29 The graph shows the results of the survival rate detection in the mouse infection model;

[0054] Figure 30 The graph shows the results of mouse treatment survival rate detection;

[0055] Figure 31 The graph shows the bacterial load detection results of the heart (A), liver (B), spleen (C), lung (D), and kidney (E) of mice;

[0056] Figure 32 The results of histopathological analysis of organs (heart, liver, spleen, lung, kidney) in a mouse infection model (A) and H&E staining results (B) (×400). Detailed Implementation

[0057] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0058] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included within the scope of this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0059] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of this invention.

[0060] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0061] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0062] The amino acid sequence of the antimicrobial protein CB6-C used in the following examples is shown in SEQ ID NO.1.

[0063] Example 1: Preparation of CB6-C-NLCs

[0064] Weigh 40 mg of antimicrobial protein CB6-C, 50 mg of lecithin, and 10 mg of cholesterol. Add 2.5 mL of anhydrous ethanol and 0.5 mL of dichloromethane to a centrifuge tube (10 mL). Use an ultrasonic cell disruptor to sonicate for 10 min at 200 W to completely dissolve the liposomes. Use a rotary evaporator to evaporate the organic reagents at 40 °C to form a thin film inside the flask. Add 1.0 mL of sterile water to the flask and hydrate by rotation for 45 min. Sonicate the resulting solution with a probe at 200 W for 10 min. Then, use an Avanti liposome extruder to extrude NLCs sequentially through 0.4 μm, 0.2 μm, and 0.1 μm polycarbonate membranes 20 times each to obtain CB6-C-NLCs liposomes with a particle size of less than 200 nm and good dispersibility.

[0065] Example 2: Optimization of CB6-C-NLC preparation process using encapsulation efficiency and drug loading rate as indicators through single-factor experiments.

[0066] 1. Single-factor experimental screening of CB6-C-NLCs preparation process

[0067] Screening was conducted using the dosages of antimicrobial protein CB6-C, ethanol, dichloromethane, lecithin, and cholesterol, as well as ultrasound parameters, as single-factor experiments. The encapsulation efficiency and drug loading rate of CB6-C-NLCs were determined, and the results are shown in [Figure number missing]. Figures 1-6 The optimal preparation process parameters were obtained as follows: CB6-C 40mg, total volume of anhydrous ethanol and dichloromethane 3mL (volume ratio of anhydrous ethanol to dichloromethane 5:1), lecithin 50mg, cholesterol 10mg and sterile water 1mL, ultrasonic power 200W, ultrasonication for 10min, hydration time 40min, and the encapsulation efficiency and drug loading rate reached their peak values.

[0068] 2. The encapsulation efficiency and drug loading rate of liposomes were determined using high-speed centrifugation and ultrafiltration centrifugation, respectively. The prepared CB6-C-NLCs were first centrifuged at 12000 r / min for 30 min, and the supernatant was transferred to a 30 kD ultrafiltration tube. Then, the cells were centrifuged at 4000 r / min for 20 min, and the protein concentration in the lower layer was measured. The free protein content was calculated as M1. The total protein content in the liposomes was M2, and the mass of the lipid carrier was M0. The calculation formula is as follows:

[0069] Encapsulation efficiency: EE% = (M2 - M1) / M2 × 100%.

[0070] Drug loading rate: DL% = (M2-M1) / (M2+M0)×100%.

[0071] Example 3: Preparation of RBCM-CB6-C-NLCs and determination of erythrocyte membrane content

[0072] 1. Preparation of red blood cell membranes

[0073] Mouse whole blood was centrifuged at 2300 rpm for 10 min at 4°C, the supernatant was discarded, and the pellet was resuspended in 1×PBS buffer. Other residual blood cells were washed away to obtain red blood cells. The washed red blood cells were then placed in an ice bath in 0.25×PBS hypotonic buffer for 1 h to rupture the red blood cell membrane. The cells were then centrifuged at 12000 rpm for 30 min at 4°C to remove the hemoglobin suspension. This process was repeated three times to obtain the final red blood cell membrane. The red blood cell membrane suspension was sonicated and then passed through 0.4 μm, 0.2 μm, and 0.1 μm polycarbonate membranes 20 times each. The membranes were then extruded using a liposome extruder to obtain uniformly sized red blood cell membranes (RBCM), which were stored at 4°C for later use. Figure 7 ).

[0074] 2. Preparation of RBCM-CB6-C-NLCs

[0075] Weigh 40 mg of antimicrobial protein CB6-C, 50 mg of lecithin, and 10 mg of cholesterol. Add 2.5 mL of anhydrous ethanol and 0.5 mL of dichloromethane to a centrifuge tube (10 mL). Use an ultrasonic cell disruptor to sonicate for 10 min at 200 W to completely dissolve the liposomes. Use a rotary evaporator at 40 °C to evaporate the organic reagents, forming a thin film inside the flask. Add 1.0 mL of sterile water to the flask and hydrate by rotating for 40 min to prepare liposomes. Then add red blood cell membranes to make the mass ratio of red blood cell membranes to liposomes 3:10. Continue stirring and hydrating for 60 min. Use a cell disruptor probe to sonicate in an ice bath for 3 min (1 s working, 1 s interval, 100 W). The resulting suspension is extruded through polycarbonate membranes (pore sizes of 0.4, 0.2, and 0.1 μm) 20 times to obtain RBCM-CB6-C-NLCs liposomes with a particle size of approximately 200 nm and good dispersibility.

[0076] 3. Determination of erythrocyte membrane content

[0077] The concentration of erythrocyte membrane proteins was determined using the BCA method. Different erythrocyte membrane contents may affect the encapsulation efficiency of RBCM-CB6-C-NLCs. Excessive erythrocyte membrane addition may result in unbound CB6-C-NLCs. To obtain the optimal erythrocyte membrane content, RBCM-CB6-C-NLCs were prepared using erythrocyte membranes with mass ratios of 1:10, 2:10, 3:10, and 4:10. Encapsulation efficiency and drug loading were measured, and the optimal erythrocyte membrane content was determined to be 18 mg, i.e., an erythrocyte membrane to liposome ratio of 3:10. Figure 8 ).

[0078] Example 4 Preparation of CB6-C-NLCs-MRM

[0079] Red blood cell membrane liposome protein RBCM-CB6-C-NLCs (red blood cell membrane to liposome mass ratio of 3:10) were prepared according to the method of Example 3. PBP2a monoclonal antibody (purchased from Merck Darmstadt, Germany) was added, wherein the mass ratio of PBP2a monoclonal antibody to liposome was 1:50. The mixture was sonicated in an ice-water bath using a cell disruptor probe (1 second operation, 1 second interval, 100W) for 3 minutes. The resulting solution was then passed through 0.4 μm, 0.2 μm, and 0.1 μm polycarbonate membranes 20 times each using a liposome extruder to obtain PBP2a monoclonal antibody conjugated to red blood cell membrane liposome protein CB6-C-NLCs-MRM with a particle size of approximately 200 nm and good dispersibility.

[0080] Example 5: Validation and Characterization of CB6-C-NLCs-MRM

[0081] The CB6-C-NLCs used in this example were prepared in Example 1, the RBCM-CB6-C-NLCs were prepared in Example 3 (the mass ratio of erythrocyte membrane to liposomes was 3:10), and the CB6-C-NLCs-MRM were prepared in Example 4.

[0082] 1. SDS-PAGE characterization of erythrocyte membrane proteins in CB6-C-NLCs-MRM

[0083] To evaluate the retention of erythrocyte membrane proteins during the preparation of CB6-C-NLCs-MRM, this invention employed the BCA protein quantification method and SDS-PAGE electrophoresis to determine protein content. Five experimental groups were set up: CB6-C, NCLs, CB6-C-NLCs, RBCM-CB6-C-NLCs, and CB6-C-NLCs-MRM, with the original erythrocyte membrane suspension serving as a control. The specific procedures are as follows:

[0084] Take 1 mL of each of CB6-C, NCLs, CB6-C-NLCs, RBCM-CB6-C-NLCs, and CB6-C-NLCs-MRM, centrifuge at 12000 rpm for 10 min, collect the precipitate, add membrane protein lysis buffer, lyse on ice for 30 min, centrifuge again, and collect the supernatant as the sample to be tested. Protein concentration of each group of samples is determined using the BCA method. The sample loading volume is 40 μg total protein / well, and constant voltage electrophoresis mode is used: stacking gel stage 80V, 30 min; separating gel stage 120V. After electrophoresis, stain the gel in Coomassie Brilliant Blue R-250 staining solution at room temperature for 2 h. Then rinse three times with deionized water, transfer to destaining solution (methanol:acetic acid:water = 4:1:5) for 24 h, changing the destaining solution 4 times during this period, until the background is clear and protein bands are visible. Figure 9 As shown in Figure A, to verify the integrity of erythrocyte membrane surface proteins in the nanosuspension, SDS-PAGE was used to analyze the protein composition and content of NLCs, CB6-C, CB6-C-NLCs, RBCM-CB6-C-NLCs, and CB6-C-NLCs-MRM. The results showed that only a single band of CB6-C protein was observed in CB6-C-NLCs, with no erythrocyte membrane protein bands. However, compared to CB6-C-RBC, almost all bands of CB6-C protein and erythrocyte membrane proteins were detected in the RBCM-CB6-C-NLCs and CB6-C-NLCs-MRM groups, indicating that erythrocyte membrane proteins can be completely preserved in the prepared CB6-C-NLCs-MRM material.

[0085] Western blotting assays are used to analyze specific protein markers in erythrocyte membranes, such as Band 3. Figure 9 As shown in Figure B, compared with the positive control group RBC, Band3 protein was expressed in RBCM, RBCM-CB6-C-NLCs and CB6-C-NLCs-MRM, indicating that the natural membrane proteins of erythrocyte membranes can be preserved during the preparation of RBCM-CB6-C-NLCs and CB6-C-NLCs-MRM materials.

[0086] 2. Particle size and potential characterization

[0087] The hydrodynamic diameter and surface charge of the nanoparticle formulation were determined using a nanoparticle size and Zeta potential analyzer. Each sample was measured in triplicate at 25±1℃, and the final particle size data were the arithmetic mean. The Zeta potential was measured using the same method. CB6-C-NLCs were prepared according to the optimal formulation process, and their particle size distribution and potential distribution were analyzed. Figure 10As shown, the average particle size of CB6-C-NLCs was 166.6 ± 8.1 nm, the potential was -54.07 ± 0.95 mV, and the PDI was 0.47 ± 0.14. RBCM-CB6-C-NLCs were prepared according to the optimal formulation process, and the results of their particle size distribution and potential distribution are as follows. Figure 11 As shown, the average particle size was 193.65 ± 3.45 nm, the potential was -52.5 ± 0.28 mV, and the PDI was 0.32 ± 0.04. CB6-C-NLCs-MRM were prepared according to the optimal formulation process, and the particle size distribution was analyzed as follows: Figure 12 As shown, its average particle size is 171.2±4.54nm, its potential is -51.47±0.76mV, and its PDI is 0.42±0.04.

[0088] 3. Morphological characterization

[0089] The microstructure of CB6-C-NLCs-MRM was characterized by transmission electron microscopy. After appropriate dilution of CB6-C-NLCs-MRM with deionized water, 5 μL was added dropwise onto a copper grid supported by a carbon film. The sample was then negatively stained with 2% phosphotungstic acid solution for 1 min. After drying at room temperature, the sample morphology was observed and electron micrographs were acquired under an accelerating voltage of 80 kV. The morphological observation results of CB6-C-NLCs-MRM are as follows: Figure 13 As shown in Figure A, it presents as a milky white suspension, uniformly dispersed; the transmission electron microscopy observation results are as follows. Figure 13 As shown in Figure B, CB6-C-NLCs-MRM exhibits a typical spherical vesicle structure, with uniform size and clear, complete boundaries.

[0090] 4. X-ray diffraction characterization (XRD)

[0091] Phase analysis of each sample (CB6-C, CB6-C-NLCs, RBCM-CB6-C-NLCs, CB6-C-NLCs-MRM) was performed using X-ray diffraction. After freeze-drying, the samples were ground uniformly and flattened in a sample holder. The scanning range was 10-80° (2θ), the scanning rate was 10° / min, the current was 30mA, the voltage was 40kV, and the wavelength was... The crystal form variation of each sample was analyzed by diffraction patterns with a step size of 0.02°. Figure 14 ).

[0092] 5. Fourier Transform Infrared Absorption Spectrometer (FTIR)

[0093] Molecular structure analysis of lyophilized samples was performed using Fourier transform infrared absorption spectroscopy. Each sample (CB6-C, CB6-C-NLCs, RBCM-CB6-C-NLCs, CB6-C-NLCs-MRM) was pretreated and then mixed with potassium bromide, compressed into pellets, and incubated at 400-4000 cm⁻¹. -1 Scanning was performed within the wavenumber range, with a resolution of 4 cm. -1 The scan was performed 32 times. Changes in drug molecule structure were analyzed using characteristic absorption peaks. Figure 15 ).

[0094] Example 6: In vitro drug release study

[0095] The CB6-C-NLCs used in this example were prepared in Example 1, the RBCM-CB6-C-NLCs were prepared in Example 3 (the mass ratio of erythrocyte membrane to liposomes was 3:10), and the CB6-C-NLCs-MRM were prepared in Example 4.

[0096] This invention employs dialysis to investigate the drug release behavior of nanoparticles in different media. To simulate the release pattern of CB6-C in vivo, 1 mL of CB6-C-NLC, RBCM-CB6-C-NLCs, and CB6-C-NLCs-MRM were respectively placed into 50 kDa molecular weight cutoff dialysis bags. The dialysis bags were placed in 20 mL of phosphate-buffered saline (PBS, pH 7.4) and incubated at 37°C. During incubation, 1 mL of liquid was taken from the external medium of the dialysis bag at 0.5, 1, 2, 4, 6, 8, 10, 12, 24, 48, 72, and 96 h. After each sampling, an equal volume (1 mL) of PBS buffer was immediately added to maintain a constant release volume. The protein concentration in the samples was determined using the BCA method and used as the drug release concentration. The cumulative release rate was then calculated. Figure 16 The results demonstrated the in vitro release efficiency of the three materials, CB6-C-NLCs, RBCM-CB6-C-NLCs, and CB6-C-NLCs-MRM, in PBS buffer (pH 7.4). The CB6-C-NLCs group was completely released within 48 hours, while the RBCM-CB6-C-NLCs and CB6-C-NLCs-MRM groups were completely released within 96 hours, which delayed the release time compared to the CB6-C-NLCs group.

[0097] Example 7: MIC determination of antimicrobial proteins CB6-C and CB6-C-NLCs-MRM

[0098] The CB6-C-NLCs used in this example were prepared in Example 1, the RBCM-CB6-C-NLCs were prepared in Example 3 (the mass ratio of erythrocyte membrane to liposomes was 3:10), and the CB6-C-NLCs-MRM were prepared in Example 4.

[0099] The minimum inhibitory concentrations (MICs) of CB6-C, RCM-CB6-C-NLCs, NLCs-MRM, and CB6-C-NLCs-MRM were determined using the microbroth dilution method recommended by the Clinical Laboratory Standards Committee (CLSIM) of the United States. MIC determinations were made according to CLSIM 100-S25 (2015). The specific procedures were as follows: the sample was dissolved in sterile distilled water to a concentration of 512 μg / mL, and then serially diluted 2-fold to 10 gradients using LB medium. 100 μL of each dilution was added sequentially to wells 1-10 of a 96-well plate. Meanwhile, MRSA glycerol indicator bacteria stored at -80°C were streaked, and single colonies were inoculated into LB broth and cultured at 37°C and 180 rpm until the logarithmic growth phase (OD50). 600 =0.5), adjust the bacterial concentration to 10 using LB medium. 5 CFU / mL, indicator bacteria were added to wells 1-10 containing sample dilution at a rate of 100 μL / well. In addition, 200 μL of LB broth was added to well 11 as a negative control, and 100 μL of LB broth and 100 μL of diluted MRSA indicator bacteria were added to well 12 as a positive control. After incubation at 37°C for 10 h, the absorbance at 600 nm of each well was measured using a microplate reader. This experiment was repeated three times.

[0100] The results are shown in Table 1. The MIC value of CB6-C against MRSA was 16 μg / mL. The MIC value of CB6-C liposomes + erythrocyte membrane (RBCM-CB6-C-NLCs) against MRSA was 16 μg / mL. The MIC value of empty liposomes + erythrocyte membrane + PBP2a monoclonal antibody (NLCs-MRM) against MRSA was >256 μg / mL. The MIC value of CB6-C-NLCs-MRM against MRSA was 8 μg / mL.

[0101] Table 1. MIC determination of antimicrobial proteins CB6-C and CB6-C-NLCs-MRM

[0102]

[0103] Example 8: Determination of growth curve and bactericidal curve

[0104] The CB6-C-NLCs-MRM used in this embodiment was prepared in Example 4.

[0105] MRSA was cultured in a 37°C incubator until the logarithmic growth phase, and the concentration of the MRSA culture was adjusted to 1×10⁻⁶. 5 After reaching CFU / mL, bacterial culture was added to test tubes containing the same volume of CB6-C-NLCs-MRM at different concentrations, ensuring that the CB6-C-NLCs-MRM content in the test tubes was 1×MIC, 2×MIC, and 4×MIC, respectively. The group with PBS and bacterial culture served as the control group. All samples were incubated together at 37℃ for 24 hours. Equal volumes of samples were taken at 0, 1, 3, 4, 6, 8, 10, 12, and 24 hours for OD measurement using a UV spectrophotometer. 600 The experiment was repeated three times, and growth curves were plotted. 100 μL samples were collected at 0, 1, 3, 4, 6, 8, 10, 12, and 24 hours for colony counting. The experiment was repeated three times, and sterilization curves were plotted. The growth curves of MRSA with different concentrations of CB6-C-NLCs-MRM are shown below. Figure 17 As shown in Figure A, compared with the control group, 2×MIC and 4×MIC could completely inhibit the growth of MRSA. The results of the bactericidal curve determination of different concentrations of CB6-C-NLCs-MRM against MRSA are shown in Figure A. Figure 17 As shown in Figure B, CB6-C-NLCs-MRM exhibited a significant inhibitory effect on MRSA growth with prolonged treatment time. Compared with the control group, 1×MIC CB6-C-NLCs-MRM reduced the number of MRSA colonies by approximately four orders of magnitude; 2×MIC and 4×MIC showed even more significant bactericidal effects, completely killing MRSA at 8h and 6h, respectively.

[0106] Example 9: Hemolytic Activity Study

[0107] The CB6-C-NLCs-MRM used in this embodiment were prepared according to the method in Example 4, with the mass ratio of red blood cell membrane to liposomes being 1:10, 2:10, 3:10, and 4:10, respectively.

[0108] Fresh blood was collected from healthy sheep and placed in 10 mL anticoagulant tubes. The blood was centrifuged at 3000 rpm for 15 min, washed five times with sterile PBS, and a 4% red blood cell suspension was prepared. Equal volumes of sheep blood cells were mixed with different concentrations of antimicrobial proteins CB6-C (1 μg / mL–1024 μg / mL) and CB6-C-NLCs-MRM (1 μg / mL–1024 μg / mL), respectively. The mixtures were incubated at 37°C for 1 h, centrifuged at 3000 × g for 10 min, and 100 μL of the supernatant was transferred to a 96-well plate. Triton X-100 (0.2%) and PBS were used as positive and negative controls, respectively. The absorbance of the mixture was measured at 570 nm. Each experiment was repeated three times. The hemolysis rate was calculated using the following formula:

[0109] Hemolysis rate (%) = (OD) sample -OD blank ) / (OD TritonX-100 -OD blank )×100%;

[0110] The lowest concentration that can cause hemolysis of more than 10% of red blood cells is defined as the minimum hemolytic concentration (MHC10).

[0111] This invention determined the hemolytic activity of the antimicrobial proteins CB6-C and CB6-C-NLCs-MRM (erythrocyte membrane to liposome membrane mass ratios of 1:10, 2:10, 3:10, and 4:10). The results are as follows: Figure 18 As shown, compared with the CB6-C group, the hemolytic activity of CB6-C-NLCs-MRM was significantly reduced in the concentration range of 256-1024 μg / mL. Although the hemolytic activity of CB6-C-NLCs-MRM was higher than that of CB6-C in the concentration range of 1-128 μg / mL, it did not exceed 10%. Further analysis showed that at the same drug dose, when the mass ratio of erythrocyte membrane to liposome membrane was 3:10, the hemolytic activity of CB6-C-NLCs-MRM was lower than that of other ratio groups, and the hemolytic activity of CB6-C-NLCs-MRM (3:10) was less than 10% in the concentration range of 1-256 μg / mL. This indicates that CB6-C-NLCs-MRM (3:10) has no hemolytic activity in this concentration range.

[0112] Example 10 Cytotoxicity Study

[0113] The CB6-C-NLCs-MRM used in this embodiment was prepared in Example 4.

[0114] The toxicity assay results of CB6-C-NLCs-MRM against mouse monocyte / macrophage RAW264.7 and human embryonic kidney cells HEK293T are as follows: Figure 19 As shown, high concentrations of CB6-C-NLCs-MRM had almost no effect on the survival of RAW264.7 and HEK293T. In particular, when the concentration of the antimicrobial protein CB6-C in CB6-C-NLCs-MRM was 2048 μg / mL, the survival rates of RAW264.7 and HEK293T were both above 90%. These experimental results indicate that a certain concentration of CB6-C-NLCs-MRM has no effect on the survival rate of RAW264.7 and HEK293T.

[0115] Example 11 Acute toxicity and subtoxicity study

[0116] The CB6-C-NLCs-MRM used in this embodiment was prepared in Example 4.

[0117] To evaluate the in vivo safety of CB6-C-NLCs-MRM, we conducted an acute toxicity study of CB6-C-NLCs-MRM according to Appendix XIC of the 2020 Chinese Pharmacopoeia, Volume IV, and the "Experimental Methods and Techniques for Toxicology". LD1 (Lethal Dose 1%) refers to the dose of a drug or chemical substance required to cause death in 1% of experimental animals, usually expressed in milligrams per kilogram of body weight (mg / kg). This is an indicator used in toxicology to describe the toxicity intensity of a substance. In this experiment, we selected 3 / 1 LD1 and 6 / 1 LD1 doses for toxicity studies. Forty Kunming mice aged 6-8 weeks and weighing 25±2g were acclimatized for one week in an environment with 12 hours of light and 12 hours of darkness per day at a temperature of 20-25℃ and a relative humidity of 60%-80%. The mice were then randomly divided into four groups (n=10 per group, 5 males and 5 females): experimental groups (20 mg / kg CB6-C, 15 mg / kg CB6-C-NCLs-MRM, 30 mg / kg CB6-C-NLCs-MRM, concentrations based on the loaded drug concentration) and a negative control group (injected with an equal volume of PBS). The mice were administered the drugs via intraperitoneal injection for 14 and 45 consecutive days, respectively. During the experiment, the mortality rate, general condition, clinical signs, and physical appearance of all mice were observed at 8-hour intervals before the experiment and throughout the drug administration period. The daily food and water consumption of the mice was measured before the start of drug administration and then every 2 days thereafter. Mice were euthanized under isoflurane anesthesia on days 15 and 46. All mice were fasted for at least 18 hours before autopsy. Blood was collected from the orbital cavity and placed in a vacuum anticoagulated blood vessel containing EDTA. The vessel was gently inverted several times. Three mice were collected from each group. The blood was then mixed and used for hematological index detection, serum biochemical analysis, and histopathological H&E staining analysis.

[0118] After 14 consecutive days of intraperitoneal injection of different doses of CB6-C and CB6-C-NLCs-MRM, compared with the PBS group, no mice in the CB6-C group or the CB6-C-NLCs-MRM group died, and there were no changes in the mice's fur, respiration, reflexes, or other behaviors, indicating no signs of poisoning. Figure 20As shown in Table A, the trends in body weight, food intake, and water consumption in mice at different concentrations of CB6-C-NLCs-MRM and the PBS group were generally consistent across the groups, indicating that 30 mg / kg CB6-C-NLCs-MRM did not significantly affect the normal physiological state of the mice. Serum biochemical indicators of the mice are shown in Table 2. Compared with the PBS group, all serum biochemical indicators in the CB6-C-NLCs-MRM group were within the normal reference range. Complete blood count indicators of the mice are shown in Table 3. Compared with the PBS control group, there were no differences in white blood cell and red blood cell counts in the CB6-C-NLCs-MRM group, all within the normal reference range, indicating that 30 mg / kg CB6-C-NLCs-MRM did not affect any of the complete blood count indicators in the mice. Figure 21 As shown in Figure A, compared with the PBS control group, no abnormalities were observed in the size, color, shape, consistency, or other characteristics of organs such as the heart, liver, spleen, lungs, and kidneys in the CB6-C and CB6-C-NCLs-MRM treatment groups. The results of H&E staining are as follows... Figure 21 As shown in Figure B, compared with the PBS control group, there were no significant changes in tissues and organs in the CB6-C and CB6-C-NLCs-MRM treatment groups, and no edema or degeneration was observed.

[0119] Table 2. Detection of serum biochemical indicators for acute toxicity in mice.

[0120]

[0121] Table 3. Detection of blood routine indicators for acute toxicity in mice.

[0122]

[0123] Mice were intraperitoneally injected daily with 20 mg / kg CB6-C, 15 mg / kg CB6-C-NCLs-MRM, and 30 mg / kg CB6-C-NCLs-MRM for 45 days. The complete blood count (CBC) parameters of the mice were then measured using an automated hematology analyzer, as shown in Table 5. Compared with the PBS control group, there were no differences in the CBC and CB6-C-NCLs-MRM administration groups, and all parameters were within the normal reference range. This indicates that a certain dose of CB6-C-NCLs-MRM did not produce hematological toxicity in mice. Furthermore, the serum biochemical parameters in Table 4 show that the changes in serum biochemical parameters in the CB6-C-NCLs-MRM administration groups were all within the normal reference range.

[0124] Mice were administered CB6-C, CB6-C-NCLs-MRM, and CB6-C-NCLs-MRM via intraperitoneal injection for 45 days. Compared to the PBS control group, there was essentially no difference in body weight gain between the CB6-C and CB6-C-NCLs-MRM groups. Figure 22 (A). The food and water intake of mice in the CB6-C-NCLs-MRM treatment group was as follows: Figure 22 As shown in Figures B and C, there was no difference between the mice in the PBS control group and the control group, indicating that administration of CB6-C and CB6-C-NLCs-MRM to the mice did not affect their food and water intake. Observations of the major organs (heart, liver, spleen, lungs, and kidneys) in the treatment group showed the following results: Figure 23 As shown in Figure A, the shape, size, and color of all organs in the mice were normal, and no abnormalities such as congestion, edema, or necrosis were observed. Furthermore, after H&E staining of the organs in the drug-treated mice, the results under a microscope are as follows: Figure 23 As shown in Figure B, the tissue structure of each organ in the mice treated with the PBS control group showed no significant difference compared with that in the PBS control group, and no cell or tissue degeneration was observed.

[0125] Table 4. Detection of serum biochemical indicators of subchronic toxicity in mice

[0126]

[0127] Table 5. Detection of routine blood indicators in mice with subchronic disease.

[0128]

[0129]

[0130] Example 12 Macrophage uptake

[0131] The CB6-C-NLCs used in this example were prepared in Example 1, the RBCM-CB6-C-NLCs were prepared in Example 3 (the mass ratio of erythrocyte membrane to liposomes was 3:10), and the CB6-C-NLCs-MRM were prepared in Example 4.

[0132] Take 40 μL of 1 mM DiD stock solution and add it to PBS, CB6-C-NLCs, RBCM-CB6-C-NLCs, and CB6-C-NLCs-MRM solutions respectively. Dialyze in the dark for 24 h to obtain DiD-labeled nanoliposomes.

[0133] RAW264.7 cells were seeded in laser confocal microplates (2 × 10⁻⁶ cells / mL). 4Cells / well were attached to the culture vessel, washed with PBS, and then 0.2 mL of DiD-labeled nanoparticle suspension (Free DiD, CB6-C-NLCs, RBCM-CB6-C-NLCs, CB6-C-NLCs-MRM) and 1.8 mL of cell culture medium were added. The mixture was gently shaken and incubated in a 5% CO2 incubator at 37°C for 30 min. PBS alone with DiD dye served as the control group (Free DiD), and was incubated at 37°C for 30 min. PBS + DiD served as the Free DiD control group. After incubation, cells were fixed with 4% paraformaldehyde, stained with DAPI, mounted, and observed using a laser confocal microscope. DAPI excitation / emission wavelengths were 360 / 460 nm (blue), and DiD wavelengths were 664 / 665 nm (red). Results are as follows: Figure 24 As shown in Figure A, both the Free DiD and CB6-C-NLCs groups without erythrocyte membranes exhibited intracellular fluorescence fusion, while the RBCM-CB6-C-NLCs and CB6-C-NLCs-MRM groups showed no intracellular fluorescence fusion after the addition of erythrocyte membranes, suggesting that it can reduce cellular uptake. Flow cytometry was used to quantitatively analyze the uptake of each group of nanoliposome suspensions by RAW264.7 cells, ensuring that at least 1×10⁻⁶ samples were collected for each assay. 4 The number of cells was determined, and the experiment was repeated three times to ensure the reliability of the results. Flow cytometry analysis further showed that the introduction of the erythrocyte membrane can effectively evade macrophage uptake. Figure 24 (B)

[0134] Example 13: In vitro targeted MRSA uptake study using laser confocal microscopy.

[0135] The CB6-C-NLCs used in this example were prepared in Example 1, the RBCM-CB6-C-NLCs were prepared in Example 3 (the mass ratio of erythrocyte membrane to liposomes was 3:10), and the CB6-C-NLCs-MRM were prepared in Example 4.

[0136] MRSA was cultured at 37°C until the logarithmic growth phase, and the bacterial concentration was adjusted to 1×10⁻⁶. 8CFU / mL, 1 mL of bacterial culture was centrifuged at 8000 rpm for 10 min at 4℃, and washed three times with PBS. Then, 0.2 mL of DiD-labeled nanoliposomes (Free DiD, CB6-C-NLCs, RBCM-CB6-C-NLCs, CB6-C-NLCs-MRM) and 1.8 mL of LB medium were added, and the mixture was incubated at 37℃ with shaking for 1 h. After incubation, the free nanoparticles were removed by centrifugation, and the cells were washed three times with PBS. Diluted DAPI dye was added and stained in the dark for 20 min, followed by two more centrifugations and washes. Finally, the MRSA cells were resuspended in 300 μL of PBS. The uptake of each group of nanoliposomes in MRSA was observed using a laser confocal microscope (DAPI excitation / emission: 360 / 460 nm, DiD excitation / emission: 664 / 665 nm). The results are shown below. Figure 25 As shown in Figure A, within 1 hour, MRSA showed extremely low uptake of Free DiD, CB6-C-NLCs, and RBCM-CB6-C-NLCs without PBP2a monoclonal antibody, with almost no red fluorescence signal observed in DiD-labeled NLCs. However, after the addition of PBP2a monoclonal antibody, the red fluorescence signal of DiD-labeled CB6-C-NLCs-MRM was attached to the blue fluorescence signal of DAPI-labeled MRSA. This difference indicates that MRSA can better target and uptake CB6-C-NLCs-MRM containing PBP2a monoclonal antibody. Flow cytometry was used to quantitatively analyze the uptake of each group of nanoliposome suspensions by MRSA, ensuring at least 1×10⁻⁶ samples were collected for each assay. 5 The number of MRSA colonies was counted, and the experiment was repeated three times to ensure the reliability of the results. Flow cytometry analysis also showed that MRSA can effectively target and take up CB6-C-NLCs-MRM ( Figure 25 (B). The above results indicate that CB6-C-NLCs-MRM has a good targeting effect on MRSA.

[0137] Example 14 In vivo targeting study in mice

[0138] The CB6-C-NLCs used in this example were prepared in Example 1, the RBCM-CB6-C-NLCs were prepared in Example 3 (the mass ratio of erythrocyte membrane to liposomes was 3:10), and the CB6-C-NLCs-MRM were prepared in Example 4.

[0139] DiD-labeled nanoliposome suspensions (CB6-C-NLCs, RBCM-CB6-C-NLCs, CB6-C-NLCs-MRM) were used to investigate the biodistribution and targeting of the prepared CB6-C-NLCs and CB6-C-NLCs-MRM in mice. DiD-labeled CB6-C-NLCs, RBCM-CB6-C-NLCs, and CB6-C-NLCs-MRM were intraperitoneally injected into mice infected with MRSA at a dose of 20 mg / kg. The enrichment of MRSA in various organs of mice was detected using a mouse in vivo imaging system. Animal grouping, drug administration, and imaging: Mice successfully infected with MRSA were randomly divided into 5 groups of 15 mice each. Each mouse was injected intraperitoneally with PBS, Free DiD, CB6-C-NLCs, RBCM-CB6-C-NLCs, or CB6-C-NLCs-MRM (200 μL per mouse). Mice were then anesthetized with 3% isoflurane and placed in a small animal in vivo optical imaging system for analysis. The distribution of the nanoliposome suspension in mice was observed at different time points (0, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, and 48 h) using the mouse in vivo imaging system. Mice were sacrificed at 4 h, 8 h, 12 h, 24 h, and 48 h, and the heart, liver, spleen, lung, and kidney were immediately removed for in vitro imaging and analysis using the same method. In fluorescence mode, the excitation wavelength was selected as 644 nm, and the emission wavelength as 665 nm. The total radiative efficiency was measured using Living Image software.

[0140] like Figure 26 As shown, the fluorescence signal in the PBS group was weaker, with almost no signal at the same fluorescence level. This indicates that mouse autofluorescence and normal PBS do not interfere with the measurement of liposome fluorescence. Figure 27 Fluorescence distribution in mouse organs and Figure 28Semi-quantitative fluorescence IVIS analysis of AC in mice showed that different tissues and organs of mice infected with MRSA exhibited different fluorescence distributions after treatment with various drugs. The liver and spleen showed the strongest fluorescence intensity, which is speculated to be because the liver is the largest clearance organ, exhibiting aggregation of nanoliposome suspensions. Specifically, the CB6-C-NLCs group showed fluorescent signals in the liver, spleen, and lungs at 4 hours, while the RBCM-CB6-C-NLCs and CB6-C-NLCs-MRM groups showed no fluorescent signals in the liver, spleen, and lungs, indicating that the introduction of erythrocyte membranes can effectively prolong the circulation time of drugs in vivo. At 8h and 12h, the fluorescence intensity of CB6-C-NLCs increased, and the liver and spleen of the RBCM-CB6-C-NLCs group began to aggregate fluorescence, while the CB6-C-NLCs-MRM group showed no fluorescence. This may be because CB6-C-NLCs-MRM contains a PBP2a monoclonal antibody, which targets MRSA during its entry into various organs, preventing direct entry and resulting in weaker fluorescence intensity in these organs. At 24h and 48h, the liver of the CB6-C-NLCs-MRM group began to fluoresce. This may be because after 24h, the liver of the mice in the CB6-C-NLCs-MRM group contained the highest amount of MRSA, and due to its MRSA-targeting activity, it could bind to MRSA in the liver, leading to enhanced liver fluorescence. Furthermore, as... Figure 28 As shown in the DF, compared with the CB6-C-NLCs and RBCM-CB6-C-NLCs groups, the CB6-C-NLCs-MRM group can effectively reduce the bacterial load in various organs of mice, further verifying that CB6-C-NLCs-MRM has the effect of targeted clearance of MRSA.

[0141] Example 15: Experiment on the therapeutic effect of CB6-C-NLCs-MRM on mice

[0142] The CB6-C-NLCs-MRM used in this embodiment was prepared in Example 4.

[0143] Preliminary experimental results are as follows Figure 29 As shown, when mice were injected intraperitoneally with 250 μL of 5 × 10 9 When MRSA was administered at CFU / mL, the survival rate of mice within 48 hours was 10%. When mice were intraperitoneally injected with 250 μL of 1×10... 10 When MRSA was administered at CFU / mL, the survival rate of mice was 10% within 36 hours, and all mice died within 48 hours. Therefore, 250 μL of 5 × 10⁻⁶ CFU / mL MRSA was used. 9 The MRSA dose of CFU / mL was used to subsequently construct a mouse model of acute peritonitis infection.

[0144] Mice were injected intraperitoneally with 5×109 CFU / mL MRSA bacterial suspension caused 100% mortality in mice within 48 hours. A mouse treatment model was established: Seventy 4-week-old Kunming mice were randomly divided into 7 groups (n=10 per group, 5 males and 5 females) and fed under the above conditions for one week. The experiment included a blank control group (PBS), a positive control group (MRSA + 20 mg / kg vancomycin), a negative PBS control group (MRSA + PBS), and different concentrations of drug treatment groups (MRSA + 20 mg / kg CB6-C, MRSA + 10 mg / kg CB6-C-NLCs-MRM, MRSA + 20 mg / kg CB6-C-NLCs-MRM, MRSA + 30 mg / kg CB6-C-NLCs-MRM). Except for the blank control group, all mice were injected with 250 μL (5 × 10 ml) in the right abdomen. 9 MRSA bacterial suspension at CFU / mL was administered. One hour later, mice in the positive control group and drug treatment group were treated by injecting vancomycin, CB6-C, and different concentrations of CB6-C-NLCs-MRM into the left abdominal paw, respectively. Mice were observed for 48 hours, and their survival was recorded. After 48 hours, the mice were euthanized, and heart, liver, spleen, lung, and kidney tissues were collected for organ bacterial load, inflammatory cell infiltration, and histopathological examination.

[0145] like Figure 30 As shown, compared with the PBS control group, the survival rate of mice treated with 30 mg / kg CB6-C-NLCs-MRM was 90% within 48 hours, and the survival rate of mice treated with 20 mg / kg antimicrobial protein CB6-C was 60% within 48 hours. These results indicate that different doses of CB6-C-NLCs-MRM have significant therapeutic effects on mice infected with MRSA.

[0146] The bacterial load in tissues and organs of mice infected with MRSA after treatment with CB6-C, vancomycin, and different doses of CB6-C-NLCs-MRM is as follows: Figure 31 As shown, compared with the PBS group, treatment with 30 mg / kg CB6-C-NLCs-MRM reduced bacterial load in the liver and spleen of mice by 5 orders of magnitude, and in the heart, lungs, and kidneys by 3 orders of magnitude. Furthermore, the bacterial load in all organs of mice treated with 30 mg / kg CB6-C-NLCs-MRM was significantly lower than that in the CB6-C protein treatment group and the vancomycin treatment group. These results indicate that CB6-C-NLCs-MRM significantly reduces bacterial load in the organs of infected mice compared to CB6-C protein treatment alone, and its therapeutic effect is superior to that of the vancomycin treatment group. Figure 32As shown, treatment with CB6, vancomycin, and different concentrations of CB6-C-NLCs-MRM significantly alleviated the pathological damage to various tissues and organs in mice infected with MRSA. These experimental results indicate that CB6-C-NLCs-MRM can effectively reduce organ pathological damage induced by MRSA infection in mice and has a significant therapeutic effect on organ damage in mice.

[0147] The CB6-C-NLCs-MRM nanomedicine system disclosed in this invention is expected to become a new approach for targeted therapy of MRSA, providing new hope for the treatment of MRSA infection.

[0148] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing an antibacterial protein nano-drug delivery system, characterized in that, Includes the following steps: The liposomes encapsulating the antimicrobial protein CB6-C were mixed with the erythrocyte membrane to obtain RBCM-CB6-C-NLCs liposomes; The RBCM-CB6-C-NLCs liposomes were mixed with PBP2a monoclonal antibody to obtain the antibacterial protein nano-delivery system. The amino acid sequence of the antimicrobial protein CB6-C is shown in SEQ ID NO.

1.

2. The preparation method according to claim 1, characterized in that, The method for preparing the liposomes encapsulating the antimicrobial protein CB6-C includes the following steps: The antimicrobial protein CB6-C, lecithin, and cholesterol are dissolved in a solvent. After the solvent is evaporated, water is added for hydration to obtain liposomes encapsulating the antimicrobial protein CB6-C.

3. The preparation method according to claim 2, characterized in that, The mass ratio of the antimicrobial protein CB6-C, the lecithin, and the cholesterol is 4:5:

1.

4. The preparation method according to claim 2, characterized in that, The solvent includes anhydrous ethanol and dichloromethane; and / or The volume ratio of the anhydrous ethanol to the dichloromethane is 5:

1.

5. The preparation method according to claim 1, characterized in that, The mass ratio of the red blood cell membrane to the liposomes encapsulating the antimicrobial protein CB6-C is (1-4):

10.

6. The preparation method according to claim 5, characterized in that, The mass ratio of the red blood cell membrane to the liposomes encapsulating the antimicrobial protein CB6-C is 3:

10.

7. An antibacterial protein nano-drug delivery system prepared by the preparation method according to any one of claims 1-6.

8. The application of the antimicrobial protein nano-drug delivery system as described in claim 7 in the preparation of antimicrobial drugs.

9. An antibacterial drug, characterized in that, Including the antimicrobial protein nano-delivery system as described in claim 7.

10. The antibacterial drug according to claim 9, characterized in that, The antimicrobial drug also includes pharmaceutically acceptable excipients.

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