A nitric oxide donor type ginger exosome fusion liposome nano drug delivery system and a preparation method and application thereof

Abstract

The application discloses a kind of nitric oxide donor type raw ginger exosome fusion liposome nanodelivery system and its preparation method and application, the nanodelivery system uses super flow control chip technology to nitric oxide precursor, two stearyl phosphatidylcholine, cholesterol, tobramycin, self-assembly, and then prepared by extrusion method and raw ginger exosome membrane fusion.The drug delivery system has a significant breakthrough in mucus barrier and antibacterial efficacy, and can be applied to inhalation therapy for pseudomonas aeruginosa lung infection.The negative surface of the drug delivery system and the electrostatic repulsion of mucin in mucus, mPEG-PNTC generates NO due to GSH responsiveness, which has a gas motor effect, enhancing the effect of breaking through the airway mucus barrier, while the NO generated by mPEG-PNTC, through nitrosylation and oxidative stress, destroys bacterial biofilm, interferes with bacterial iron metabolism, and synergistically kills bacteria and destroys biofilm.The present application has the advantages of improving drug delivery efficiency and high-efficiency bactericidal function, and has broad clinical application prospects and development value in the field of bacterial lung infection treatment.

Description

Technical Field

[0001] This invention belongs to the field of inhaled drug formulations, specifically relating to a nitric oxide donor-type ginger exosome fusion liposome nanodelivery system and its preparation method and application. Background Technology

[0002] Pseudomonas aeruginosa is a common opportunistic pathogen in clinical practice and a frequent cause of refractory lung infections. In 2024, the World Health Organization listed Pseudomonas aeruginosa as a high priority pathogen. Due to the increasing multidrug resistance of Pseudomonas aeruginosa, antibiotic treatment has become extremely difficult, necessitating the search for new anti-infective strategies.

[0003] Tobramycin (TOB) is an effective drug for treating Pseudomonas aeruginosa lung infections. However, its intravenous administration has poor penetration into lung tissue, resulting in low drug concentrations in the lungs; simultaneously, systemic administration may cause adverse reactions such as nephrotoxicity and ototoxicity. Therefore, nebulized inhalation is often used clinically to increase the local drug concentration in the respiratory tract. It is important to note that the respiratory tract is a complex and dynamic physiological environment, and its mucus layer may hinder the effective delivery of drugs to the alveolar region. This leads to insufficient antibiotic exposure near the Pseudomonas aeruginosa biofilm, resulting in decreased efficacy against Pseudomonas aeruginosa after repeated use.

[0004] Therefore, overcoming the problem of low lung concentrations and numerous adverse reactions of tobramycin (TOB) after intravenous administration has become a pressing technical challenge in this field. Summary of the Invention

[0005] Purpose of the invention: To address the problems existing in the current intravenous TOB treatment of Pseudomonas aeruginosa, this invention designs an inhaled nano-drug delivery system for treating Pseudomonas aeruginosa lung infections.

[0006] Technical solution: To solve the above problems, the technical solution adopted by the present invention is as follows: A nitric oxide donor-type ginger exosome fusion liposome nanoparticle drug delivery system is characterized by the use of superfluidic chip technology to self-assemble distearate phosphatidylcholine, cholesterol, tobramycin, and mPEG-PNTC into liposome nanoparticles, which are then fused with ginger exosome membranes by extrusion.

[0007] The nano-drug delivery system is characterized in that the molar ratio of DSPC, cholesterol, and mPEG-PNTC is 5~7:2~4:1.

[0008] The nano-drug delivery system is characterized in that the mass ratio of protein to liposome nanoparticles in the ginger exosomes is 1:5.

[0009] The nano-drug delivery system is characterized by the following technical parameters of the ultrafluidic chip: total flow rate of 2 mL, TFR of 1 mL / min, FFR of 4:1, and temperature of 25℃.

[0010] The nano-drug delivery system is characterized in that the mPEG-PNTC is prepared according to the following steps: under vacuum conditions, the ester cyclic carbonate monomer and methoxy polyethylene glycol are co-dissolved in anhydrous dichloromethane, the catalyst diphenylphosphine is added dropwise, and the reaction is stirred in an oil bath at 40°C. After the reaction is completed, mPEG-PNTC is obtained by post-treatment.

[0011] The nano-drug delivery system is characterized in that the ginger exosomes are prepared according to the following steps: (i) Wash the fresh ginger and juice it using a juicer; (ii) Transfer the freshly squeezed ginger juice into a centrifuge tube and centrifuge at 4℃ and 1000×g for 10 min, 4℃ and 3000×g for 20 min, and 4℃ and 10000×g for 40 min in sequence. (iii) Collect the supernatant and filter it sequentially through 0.8 μm, 0.45 μm and 0.22 μm filter heads; (iv) Add 10% w / v PEG8000 to the filtered supernatant, mix well and let stand overnight at 4°C; (v) Centrifuge at 4℃ and 8000×g for 30 min; (vi) Collect the precipitate, add an appropriate volume of PBS buffer to resuspend and mix well; (vii) Use 100 kDa ultrafiltration centrifuge tubes and centrifuge at 4℃ and 4000×g for 10 min. Add PBS buffer after each centrifugation and repeat the centrifugation 3 times. (ⅷ) After filtration through a 0.22 μm filter, ginger exosomes are obtained.

[0012] The method for preparing the nano-drug delivery system is characterized by the following steps: (i) Using ethanol as the organic solvent, dissolve DSPC and cholesterol, and using acetonitrile as the organic solvent, dissolve mPEG-PNTC. Prepare an ethanol solution with a total lipid concentration of 10 mg / mL at a molar ratio of 5~7:2~4:1, and sonicate for 2 min. Then add 3 times the volume of ethanol and sonicate again for 3~5 min. Use a syringe to draw up the sonicated polymer ethanol solution and PBS buffer containing an appropriate amount of tobramycin, load them into the ultrafluidic chip, set the total flow rate to 2 mL, TFR to 1 mL / min, FFR to 4:1, and temperature to 25℃, start the instrument, and collect liposome nanoparticles in a 15 mL centrifuge tube. Place the collected solution in a dialysis bag with a molecular weight cutoff of 1000 Da, and use PB solution as the dialysis medium to remove organic reagents and unencapsulated drugs. (ii) The protein in ginger exosomes was mixed with liposome nanoparticles at a mass ratio of 1:5, vortexed for 2 min, and sonicated for 2 min; and extruded repeatedly for 30 times with 400 nm and 200 nm polycarbonate membranes respectively to obtain the target nano-drug delivery system.

[0013] The application of the described nano-drug delivery system in the preparation of drugs for treating Pseudomonas aeruginosa infections.

[0014] The above-mentioned nano-drug delivery system is used in the preparation of medical materials for the treatment of bacterial biofilm-associated infections.

[0015] Nitric oxide (NO), as an important gaseous signaling molecule, has shown great potential in various pathophysiological processes such as cardiovascular disease treatment, anti-tumor therapy, and bacterial inhibition. NO not only has a direct bactericidal effect at high concentrations, but also effectively inhibits bacterial adhesion and disperses biofilms at low, non-toxic concentrations, thereby reducing bacterial resistance to antibiotic treatment. mPEG-PNTC is a biodegradable polymeric nitric oxide (NO) donor precursor that has shown important application value in tumor immunotherapy. The NO it releases can not only directly kill tumor cells, but also has significant immunomodulatory effects, including inducing immunogenic cell death (ICD), promoting dendritic cell maturation, and T cell infiltration. Furthermore, NO can downregulate the expression of PD-L1 in tumor cells and induce phenotypic transformation in M2 macrophages, thereby effectively reshaping the tumor immunosuppressive microenvironment (https: / / doi.org / 10.1016 / j.nantod.2022.101381).

[0016] Extracellular vesicles are membrane-bound nanoscale vesicles containing lipids, proteins, and nucleic acids, and can be released by almost all living cells. Plants also contain extracellular vesicles; Regemte et al. first isolated EVs from sunflower seeds in 2009. Subsequently, scholars isolated extracellular vesicles from different plants, discovering that they possess biomolecular structures similar to mammalian exosomes in terms of size (50-200 nm) and biological carriers (lipids, proteins, and small RNAs), collectively termed exosome-like nanoparticles. In recent years, plant exosomes have been widely studied due to their high yield, low cost, low immunogenicity, and multifunctionality. Among them, ginger exosomes exhibit multiple effects, including anti-inflammatory, anticancer, anti-infective, and drug delivery. Studies have found that ginger exosomes endow nanoparticles with good biocompatibility and long blood circulation, ensuring effective accumulation of nanoparticles at the site of infection. Ginger exosomes promote the absorption of nanoparticles by bacteria, generating strong interactions with bacteria and enhancing bactericidal effects. Previous studies have shown that low-dose ginger extract has effective antibacterial and anti-biofilm activity against multidrug-resistant Pseudomonas aeruginosa, suggesting the potential value of ginger in treating Pseudomonas aeruginosa infections.

[0017] Beneficial effects: 1. This invention innovatively combines superfluidic chip technology with membrane fusion technology to construct a novel nano-drug delivery system. Its core breakthrough lies in the first-time synergistic introduction of the nitric oxide (NO) precursor mPEG-PNTC with plant exosomes into a liposome system, and the subsequent fabrication of a composite nanocarrier suitable for nebulized inhalation using a membrane fusion strategy. This drug delivery system not only significantly overcomes the mucus barrier but also exhibits potent antibacterial efficacy.

[0018] Among these, ginger exosomes significantly enhanced the mucus penetration ability of the nano-drug delivery system and endowed it with excellent biocompatibility. The negatively charged surface of this drug delivery system can generate electrostatic repulsion with mucins in the mucus, further promoting its diffusion in the mucus layer. Simultaneously, mPEG-PNTC generates NO in response to glutathione (GSH), and its gas motor effect can significantly enhance the ability of the nanocarrier to overcome the airway mucus barrier. Furthermore, the NO generated by mPEG-PNTC can disrupt bacterial biofilm structure through nitrosation and oxidative stress, interfere with bacterial iron metabolism, and synergistically exert a bactericidal effect with tobramycin, thereby achieving highly efficient antibacterial and biofilm clearance.

[0019] This system achieves potent therapeutic effects against bacterial lung infections by utilizing a novel synergistic mechanism involving NO precursor, ginger exosomes, and tobramycin, significantly improving drug delivery efficiency. This invention provides a novel treatment strategy for clinical use that combines high delivery efficiency with superior antibacterial capabilities, demonstrating significant application prospects and development value.

[0020] 2. The preparation conditions of the present invention are mild, the operation is simple, and it is suitable for large-scale production.

[0021] 3. The nano-drug delivery system of this invention can be used as an antibacterial drug via nebulized inhalation, effectively fighting infection, inhibiting and destroying biofilm growth, and reducing infection-related inflammatory responses, showing great clinical application potential in the treatment of pulmonary bacterial infections. Attached Figure Description

[0022] Figure 1. Proton NMR spectrum of PEG-PNTC in Example 1; Figure 2 Particle size distribution diagram and transmission electron microscope image of GELN@NTC@LIP@TOB in Example 2; Figure 3 Release spectrum of NO from the nano-drug delivery system in Example 3; Figure 4 Example 4 shows the accumulation of the nano-drug delivery system in the lungs, where A: in vivo imaging, B: ex vivo imaging; Figure 5 1. In vitro antibacterial spectrum (A) and statistical results (B) of the nano-drug delivery system in Example 5; Figure 6 Anti-biofilm spectrum (A) and statistical results (B) of the nano-drug delivery system in Example 6; Figure 7 Pathological morphology of lung tissue from the nano-drug delivery system in Example 7. Detailed Implementation

[0023] The technical solution of the present invention will be further described below with reference to the embodiments. The test materials used in the embodiments can all be obtained through conventional means.

[0024] In the examples, the abbreviations of the nano-drug delivery system name are: GELN for ginger exosomes, NTC for mPEG-PNTC, LIP for liposomes, and TOB for tobramycin.

[0025] Example 1, Synthesis of mPEG-PNTC: The synthetic route and process are as follows: .

[0026] The ester cyclic carbonate monomer (NTC) was pre-vacuumed, transferred into a glove box, and co-dissolved with methoxy polyethylene glycol 2000 (Mn=2000) in anhydrous dichloromethane. 2-3 drops of diphenylphosphine (DPP) catalyst were added, the reactor was sealed and transferred out of the glove box, stirred overnight in an oil bath at 40°C, dialyzed, and vacuum dried to obtain the product.

[0027] Result: As Figure 1As shown, the proton NMR spectrum of PEG-PNTC confirms that the present invention correctly synthesizes mPEG-PNTC.

[0028] Example 2, Preparation of the GELN@NTC@LIP@TOB nanodelivery system: 1. Preparation of liposome nanoparticles NTC@LIP@TOB: DSPC and cholesterol were dissolved in ethanol as the organic solvent, and mPEG-PNTC was dissolved in acetonitrile as the organic solvent. The solutions were prepared in a 6:3:1 ratio, resulting in a total lipid concentration of 10 mg / ml. The solutions were sonicated for 2 min, followed by the addition of three times the volume of ethanol and sonication for another 3-5 min. The sonicated ethanol solution containing the polymer and PBS (1 mg / mL TOB) were separately aspirated using syringes and loaded onto an ultrafluidic chip. The total flow rate was set to 2 ml, TFR to 1 mL / min, FFR to 4:1, and the temperature to 25°C. The instrument was started, and liposome nanoparticles were collected using 15 ml centrifuge tubes. The collected solution was then placed in a dialysis bag with a molecular weight cutoff of 1000 Da and dialyzed with PBS to remove organic reagents and unencapsulated drugs.

[0029] 2. Preparation of ginger exosomes: (i) Wash the fresh ginger and juice it using a juicer; (ii) Pour the freshly squeezed ginger juice into centrifuge tubes and centrifuge at 4°C, 1000×g for 10 min; at 4°C, 3000×g for 20 min; and at 4°C, 10000×g for 40 min. (iii) Take the supernatant and filter it sequentially using 0.8μm, 0.45μm, and 0.22μm filter heads; (iv) Add 10% (w / v) PEG8000, mix well, and let stand overnight (16h) at 4°C. (v) Centrifuge at 4℃, 8000×g for 30 min; (vi) Take the precipitate, add an appropriate volume of PBS to resuspend it, and mix well; (vii) Centrifuge 100 kDa ultrafiltration centrifuge tubes at 4°C, 4000×g for 10 min, add PBS after each centrifugation, and centrifuge 3 times; (ⅷ) Use a 0.22μm filter to obtain ginger exosomes.

[0030] 3. Preparation of the GELN@NTC@LIP@TOB nanodelivery system: The protein in ginger exosomes was mixed with liposome nanoparticles at a mass ratio of 1:5, and the mixture was vortexed for 2 minutes; then sonicated for 2 minutes; and extruded repeatedly 30 times using 400 nm and 200 nm polycarbonate membranes respectively to obtain GELN@NTC@LIP@TOB.

[0031] Result: As Figure 2 The nano-drug delivery system prepared by the above method has a particle size distribution of about 200 nm and a PD1 of 0.12.

[0032] Example 3, In vitro nitric oxide (NO) release study of the nano-drug delivery system: In vitro NO release was studied by incubation in PBS medium containing 10 mM GSH (pH 7.4).

[0033] The GELN@NTC@LIP@TOB nanoparticle drug delivery system was prepared according to the above method. The sample was placed in a constant-temperature shaker at 37 °C. 200 μL of release medium was collected at specified time points, and an equal volume of fresh medium was added. The released medium at room temperature was stained with a nitric oxide kit (Beyotime S0021S) and then analyzed using a microplate reader. This release experiment was repeated three times.

[0034] Result: As Figure 3 As shown, the nano-drug delivery system released approximately 4.43 μM of NO cumulatively after 96 hours.

[0035] Example 4, Characterization of lung accumulation of the nano-drug delivery system: Female Balb / c mice aged 7-8 weeks (22±2 g) were selected and instilled with Pseudomonas aeruginosa agarose beads (5×10⁻⁶) via tracheal instillation. 7 A mouse model of chronic Pseudomonas aeruginosa lung infection was established using CFU / mouse. Four days after modeling, GELN@NTC@LIP@TOB (2.2 mg / kg TOB) was administered to the mice via a liquid nebulizer. After administration, in vivo imaging was performed at 2 h, 3 h, 4 h, 6 h, 12 h, 24 h, and 48 h. At 24 h, some mice were euthanized and dissected to remove the main organs (heart, liver, spleen, lung, and kidney) for fluorescence imaging of the ex vivo organs.

[0036] Result: As Figure 4 As shown, compared with the LIP@TOB, GELN@LIP@TOB, and NTC@LIP@TOB groups, GELN@NTC@LIP@TOB was able to accumulate more in lung tissue at 24h, confirming that this nano-drug delivery system prolongs the accumulation time of TOB in the lungs.

[0037] Example 5, in vitro antibacterial experiment of the nano-drug delivery system: The antibacterial activity of the nanodelivery system was evaluated using a plate coating method. In this method, bacteria were cultured in LB broth and harvested at the exponential growth phase for antibacterial experiments. The bacteria were centrifuged, residual culture medium was discarded, and the bacteria were dissolved in PBS. The optical density (OD) of the bacteria at 600 nm was measured. 600 = 0.2 (equivalent to a bacterial concentration of 1×10) 7 The bacterial concentration was obtained by measuring CFU / mL, and the bacterial solution was diluted a certain factor to reach 1×10⁻⁶. 7 CFU / mL. PBS, TOB, GELN@NTC@LIP, LIP@TOB, GELN@LIP@TOB, NTC@LIP@TOB, and GELN@NTC@LIP@TOB groups were respectively mixed with 1 ml of 1×10 7 CFU / mL Pseudomonas aeruginosa was co-cultured for 4 h. Afterwards, it was centrifuged at 4000xg for 5 min, the supernatant was discarded, the bacterial pellet was washed three times with PBS, resuspended in PBS, and then serially diluted 1:10 to obtain the following bacterial suspensions (10... 1 10 2 10 3 10 4 10 5 10 6 and 10 7 100 µL of diluted bacterial suspension was evenly spread onto LB agar plates and incubated at 37 °C for 24 hours to form colony units, followed by colony counting. Representative images were collected using a digital camera. Each experiment was performed in triplicate.

[0038] Result: As Figure 5 As shown, the GELN@NTC@LIP@TOB group had the fewest bacteria, and the difference from other groups was significant. The reason is as follows: ginger exosomes and nitric oxide precursors work synergistically with tobramycin to induce ROS production, leading to bacterial death. Therefore, this group showed the best antibacterial performance.

[0039] Example 6, In vitro anti-biofilm experiment of the nano-drug delivery system: Add 1 mL of bacterial culture (1×10⁻⁶) 7CFU / mL was inoculated into 24-well plates, and then appropriate amounts of sample were added to PBS, TOB, GELN@NTC@LIP, LIP@TOB, GELN@LIP@TOB, NTC@LIP@TOB, and GELN@NTC@LIP@TOB groups, respectively, and incubated together for 4 hours. After incubation for 24 hours, the samples were washed three times with PBS, and then 0.1% (v / v) crystal violet solution was added and incubated for 1 hour. 200 µL of 95% (v / v) ethanol was added to dissolve the crystal violet. The experiment was repeated three times, and the absorbance was measured at 595 nm using a microplate reader.

[0040] Result: As Figure 6 As shown, this experiment evaluated the effects of different materials on the removal of Pseudomonas aeruginosa biofilm. The upper part of the petri dish plots shows the biofilm residue after each treatment group, while the lower part of the bar charts quantitatively analyzes the changes in bacterial biofilm quantity (OD) among different groups. 595 nm (Value). As can be seen from the 24-well plate plot, the GELN@NTC@LIP@TOB group had the least bacterial biofilm residue, which was significantly different from other groups, indicating that it had the best biofilm removal effect. The OD value of the bacterial biofilm further confirmed this trend. These results indicate that GELN and nitric oxide precursors significantly enhanced the anti-biofilm effect, and the synergistic release of NO promoted lipid peroxidation, exacerbating bacterial membrane damage, thereby maximizing biofilm removal. Therefore, GELN@NTC@LIP@TOB exhibited the best anti-biofilm performance.

[0041] Example 7, in vivo anti-infection experiment of the nano-drug delivery system: All experiments were conducted in accordance with the guidelines of the Chinese Committee for the Management and Use of Laboratory Animals. Female Balb / c mice (7-8 weeks old) were randomly assigned to 7 groups: PBS, TOB, GELN@NTC@LIP, LIP@TOB, GELN@LIP@TOB, NTC@LIP@TOB, and GELN@NTC@LIP@TOB. Pseudomonas aeruginosa agarose beads (5 × 10⁻⁶) were instilled via tracheal instillation. 7A mouse model of chronic Pseudomonas aeruginosa lung infection was established using CFU / mouse. Four days after modeling, mice were administered a liquid nebulizer via their lungs for two consecutive days (dose: 2.2 mg / kg TOB). On day 6, mice were euthanized by cervical dislocation, and both lungs were isolated. The lungs were repeatedly rinsed with sterile PBS to remove surface blood and mucus, weighed, and placed in a tissue homogenizer. 200 μL of PBS was added. The lung tissue was homogenized, and an appropriate amount of PBS was added to bring the homogenate to 1 mL. The homogenate was then pipetted and homogenized. 100 μL of the lung tissue homogenate was added to 900 μL of PBS and serially diluted 1:10 to a final volume. 1 10 2 10 3 10 4 10 5 10 6 and 10 7 Dilute 100 μL to a concentration of 10. 5 10 6 and 10 7 Dilute the solution by 1:1 and spread it evenly on an agar plate using a spreader. Then, invert the petri dish and incubate it in a bacterial incubator for 18 hours. The next day, remove the plate, observe the colony growth, and count the colonies.

[0042] like Figure 7 As shown, the GELN@NTC@LIP@TOB group exhibited the most significant antibacterial effect. In comparison, the GELN@LIP@TOB and NTC@LIP@TOB groups showed the second-best anti-infective effects.

[0043] The nitric oxide donor-type ginger exosome fusion liposome nanodelivery system constructed in this invention exhibits outstanding biomedical performance. This nanodelivery system demonstrates multiple therapeutic advantages in clinical applications: when used as an inhaled drug for pulmonary bacterial infections, it simultaneously achieves the following therapeutic effects: the negatively charged surface of the delivery system and the mucin in mucus exhibit electrostatic repulsion; mPEG-PNTC, due to its GSH-responsive NO generation, acts as a gas motor, enhancing its ability to overcome the airway mucus barrier; simultaneously, the NO generated by mPEG-PNTC disrupts bacterial biofilms through nitrosation and oxidative stress, interfering with bacterial iron metabolism, thus synergistically killing bacteria and destroying biofilms. This invention combines improved drug delivery efficiency with highly effective bactericidal function, providing an innovative solution for the clinical treatment of pulmonary bacterial infections, and has broad industrial application prospects and significant clinical translational value in the field of bacterial infection treatment.

Claims

1. A nitric oxide donor-type ginger exosome fusion liposome nanodelivery system, characterized in that, Liposome nanoparticles were prepared by self-assembling distearate phosphatidylcholine, cholesterol, tobramycin, and mPEG-PNTC using superfluidic chip technology, and then fused with ginger exosome membranes by extrusion.

2. The nano-drug delivery system according to claim 1, characterized in that, The molar ratio of DSPC, cholesterol, and mPEG-PNTC is 5~7:2~4:

1.

3. The nano-drug delivery system according to claim 1, characterized in that, The mass ratio of protein to liposome nanoparticles in the ginger exosomes is 1:

5.

4. The nano-drug delivery system according to claim 1, characterized in that, The technical parameters of the superfluid chip are: total flow rate of 2 mL, TFR of 1 mL / min, FFR of 4:1, and temperature of 25℃.

5. The nanomedicine delivery system according to claim 1, characterized in that, The mPEG-PNTC was prepared according to the following steps: Under vacuum conditions, the ester cyclic carbonate monomer and methoxy polyethylene glycol were co-dissolved in anhydrous dichloromethane, and the catalyst diphenylphosphine was added dropwise. The reaction was stirred in an oil bath at 40°C. After the reaction was completed, mPEG-PNTC was obtained through post-treatment.

6. The nano-drug delivery system according to claim 1, characterized in that, The ginger exosomes were prepared according to the following steps: (i) Wash the fresh ginger and juice it using a juicer; (ii) Transfer the freshly squeezed ginger juice into a centrifuge tube and centrifuge at 4℃ and 1000×g for 10 min, 4℃ and 3000×g for 20 min, and 4℃ and 10000×g for 40 min in sequence. (iii) Collect the supernatant and filter it sequentially through 0.8 μm, 0.45 μm and 0.22 μm filter heads; (iv) Add 10% w / v PEG8000 to the filtered supernatant, mix well and let stand overnight at 4°C; (v) Centrifuge at 4℃ and 8000×g for 30 min; (vi) Collect the precipitate, add an appropriate volume of PBS buffer to resuspend and mix well; (vii) Use 100 kDa ultrafiltration centrifuge tubes and centrifuge at 4℃ and 4000×g for 10 min. Add PBS buffer after each centrifugation and repeat the centrifugation 3 times. (ⅷ) After filtration through a 0.22 μm filter, ginger exosomes are obtained.

7. The method for preparing the nano-drug delivery system according to claim 1, characterized in that, To achieve this, follow these steps: (i) Using ethanol as the organic solvent, dissolve DSPC and cholesterol, and using acetonitrile as the organic solvent, dissolve mPEG-PNTC. Prepare an ethanol solution with a total lipid concentration of 10 mg / mL at a molar ratio of 5~7:2~4:1, and sonicate for 2 min. Then add 3 times the volume of ethanol and sonicate again for 3~5 min. Use a syringe to draw up the sonicated polymer ethanol solution and PBS buffer containing an appropriate amount of tobramycin, load them into the ultrafluidic chip, set the total flow rate to 2 mL, TFR to 1 mL / min, FFR to 4:1, and temperature to 25℃, start the instrument, and collect liposome nanoparticles in a 15 mL centrifuge tube. Place the collected solution in a dialysis bag with a molecular weight cutoff of 1000 Da, and use PB solution as the dialysis medium to remove organic reagents and unencapsulated drugs. (ii) The protein in ginger exosomes was mixed with liposome nanoparticles at a mass ratio of 1:5, vortexed for 2 min, and sonicated for 2 min; and extruded repeatedly for 30 times with 400 nm and 200 nm polycarbonate membranes respectively to obtain the target nano-drug delivery system.

8. The use of the nanodelivery system according to any one of claims 1 to 6 in the preparation of drugs for treating Pseudomonas aeruginosa infections.

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