A polystyrene nanocarrier for carrying polymyxin B and a preparation method and application thereof

Abstract

The present application relates to the technical field of biological medicine, in particular to a kind of polystin B-loaded nanoparticle and its preparation method and application, polystin B-loaded nanoparticle includes polymer nano core and macrophage membrane biomimetic layer coated on the surface of polymer nano core;The polymer nano core carries polystin B and citraconic anhydride modified polylysine;The surface of the macrophage membrane biomimetic layer is loaded with antibiofilm cyclic peptide.The nanoparticle can be used for preparing the drug for treating pseudomonas aeruginosa lung infection, and can reduce the cytotoxicity of free polystin B, and give consideration to antibacterial effect and drug safety, better meet the actual application demand.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a biomimetic polymyxin B-loaded nanoparticle, its preparation method, and its application. Background Technology

[0002] The core of treating Pseudomonas aeruginosa pulmonary infections lies in rapidly reducing the bacterial load in the lungs, alleviating inflammatory damage, and preventing recurrence. Currently, the treatment of this infection faces two major challenges: First, Pseudomonas aeruginosa can form biofilms composed of extracellular polysaccharides, proteins, and extracellular DNA. These biofilms can reduce the effective exposure of antimicrobial drugs within the bacterial flora and help bacteria evade host immune clearance. Second, polymyxin B, clinically used to treat drug-resistant Gram-negative bacterial infections, exhibits significant nephrotoxicity and neurotoxicity, limiting the safety of systemic administration. While simply increasing the dosage of polymyxin B can enhance its bactericidal effect, it can easily further increase toxic side effects. Therefore, finding a delivery system that can both intervene in biofilms and release antimicrobial drugs within the infected environment is crucial.

[0003] Pseudomonas aeruginosa aminopeptidase (PaAP) is a highly abundant secreted aminopeptidase in the biofilm matrix, involved in biofilm development and nutrient cycling. The cyclic peptide with the sequence ERWGHDFIK, upon cyclization with isopeptide bonds on the glutamate and lysine side chains, can inhibit PaAP activity, thereby intervening in Pseudomonas aeruginosa biofilms. However, existing antimicrobial systems still struggle to simultaneously achieve PaAP-related biofilm intervention, drug release in response to an acidic infection environment, and reduction of polymyxin B toxicity. Summary of the Invention

[0004] To address the aforementioned issues, this invention provides a biomimetic polymyxin B-loaded nanoparticle. This nanoparticle can be used to prepare drugs for treating Pseudomonas aeruginosa lung infections and can reduce the cytotoxicity of free polymyxin B, thus balancing antibacterial efficacy and drug safety, better meeting practical application needs.

[0005] The present invention provides a biomimetic polymyxin B-loaded nanoparticle, comprising a polymer nanocore and a macrophage membrane biomimetic layer coating the surface of the polymer nanocore; the polymer nanocore is loaded with polymyxin B and citrate anhydride-modified polylysine; and the surface of the macrophage membrane biomimetic layer is loaded with an anti-biomembrane cyclic peptide.

[0006] This invention employs a polymer nanocore loaded with polymyxin B and citrate anhydride-modified polylysine. By utilizing the charge reversal property of citrate anhydride-modified polylysine in an acidic environment, the nanoparticles expand in the acidic environment of infection, promoting the release of polymyxin B, which is beneficial to improving drug exposure and bactericidal effect at the infection site.

[0007] This invention coats a polymer nanocore with a biomimetic layer covering the macrophage membrane. The biomimetic layer can retain macrophage membrane characteristic proteins such as CD11b and CCR2, which is beneficial to improving the biocompatibility of nanoparticles and reducing the cytotoxicity of free polymyxin B.

[0008] This invention loads an anti-biofilm cyclic peptide onto the surface of a biomimetic layer of a macrophage membrane. This cyclic peptide can inhibit the activity of Pseudomonas aeruginosa aminopeptidase PaAP, thereby interfering with the biofilm of Pseudomonas aeruginosa. Together with the bactericidal effect of polymyxin B, it enhances the antibacterial effect against Pseudomonas aeruginosa.

[0009] In one embodiment, the amino acid sequence of the anti-biomembrane cyclic peptide is ERWGHDFIK.

[0010] In one embodiment, the side chain carboxyl group of the glutamic acid residue and the side chain amino group of the lysine residue in the anti-biomembrane cyclic peptide are linked by isopeptide bonds to form a cyclic structure, while retaining the N-terminal amino group and C-terminal carboxyl group of the peptide chain.

[0011] In one embodiment, the polymer nanocore further includes polylactic acid-glycolic acid copolymer (PLGA).

[0012] In one embodiment, the method for preparing the polylactic acid-glycolic acid copolymer includes the following steps: D,L-lactide and glycolide were mixed, and stannous octoate and lauryl alcohol were added. The mixture was reacted under vacuum. After the reaction was completed, the mixture was cooled, dissolved in anhydrous dichloromethane, and precipitated in anhydrous methanol. The precipitate was dried under vacuum to obtain polylactic acid-hydroxyacetic acid copolymer.

[0013] In one embodiment, the method for preparing the polylactic acid-glycolic acid copolymer includes the following steps: D,L-lactide and glycolide were mixed at a mass ratio of 1-2:1, and 0.02-0.03% of stannous octoate and 0.008-0.01% of lauryl alcohol (total mass of D,L-lactide and glycolide) were added. The mixture was reacted under vacuum at 170-180 °C for 1.5-2 h. After the reaction was completed, the mixture was cooled, dissolved in anhydrous dichloromethane, and precipitated in anhydrous methanol. The precipitate was dried under vacuum at 60-65 °C for 40-48 h to obtain polylactic acid-glycolic acid copolymer.

[0014] In one embodiment, the biomimetic polymyxin B-loaded nanoparticles have a particle size of 100~300 nm.

[0015] In one embodiment, the biomimetic polymyxin B-loaded nanoparticles have an average particle size of 172.1 nm at pH 7.4 and an average particle size of 262.1 nm at pH 5.5. This particle size range has been characterized and tested for structural stability. The nanoparticles remain relatively stable under normal physiological conditions, but undergo charge reversal and particle expansion under acidic conditions, which is beneficial for the release of polymyxin B.

[0016] In one embodiment, the biomimetic layer of the macrophage membrane is derived from the RAW264.7 macrophage membrane and retains the CD11b and CCR2 membrane proteins.

[0017] In one embodiment, the mass ratio of polymyxin B to citrate anhydride-modified polylysine is 0.5-2:1.

[0018] In one embodiment, the mass ratio of polymyxin B to citrate anhydride-modified polylysine is 1:1.

[0019] In one embodiment, the mass ratio of the macrophage membrane to polymyxin B is 0.5-2:1.

[0020] In one embodiment, the mass ratio of the macrophage membrane to polymyxin B is 1:1.

[0021] Another aspect of the present invention provides a method for preparing biomimetic polymyxin B-loaded nanoparticles, comprising the following steps: Macrophage cell membranes were extracted and subjected to sonication to obtain macrophage cell membrane nanoparticles; Anti-biomembrane cyclic peptides were mixed with macrophage membrane nanoparticles to obtain cyclic peptide-modified macrophage membrane nanoparticles. Polylysine was reacted with citrate anhydride to obtain citrate anhydride-modified polylysine. Polymyxin B was mixed with citrate anhydride-modified polylysine and then encapsulated with polylactic acid-glycolic acid copolymer to obtain a polymer nanocore loaded with polymyxin B. Cyclic peptide-modified macrophage membrane nanoparticles were mixed with the polymer nanocore and subjected to ultrasonic treatment to obtain the biomimetic polymyxin B-loaded nanoparticles.

[0022] In one embodiment, the macrophage membrane is obtained from RAW264.7 macrophages through repeated freeze-thaw cycles and differential centrifugation, and the obtained macrophage membrane is sonicated for 2-3 minutes to obtain macrophage membrane nanoparticles.

[0023] In one embodiment, the number of RAW264.7 macrophages was 2 × 10⁻⁶ when the macrophage membrane was extracted. 7 ~5×10 7The samples were subjected to three freeze-thaw cycles, and the differential centrifugation conditions were 4℃, 700×g for 10 min and 4℃, 14000×g for 30 min, respectively.

[0024] In one embodiment, the anti-biomembrane cyclic peptide is mixed with macrophage membrane nanoparticles at 20-30°C for 20-40 min, and then centrifuged at 20000-30000×g for 15-20 min to remove the free anti-biomembrane cyclic peptide.

[0025] In one embodiment, the citrate anhydride-modified polylysine is prepared by the following method: Dissolve 40-60 mg of polylysine in 2-5 mL of PBS buffer, add 80-90 mg of citrate anhydride, adjust the pH to neutral with sodium hydroxide solution, stir overnight at 20-30 °C, dialyze through a dialysis bag with a molecular weight cutoff of 500-600 Da for 72-80 h, and then freeze dry to obtain citrate anhydride-modified polylysine.

[0026] In one embodiment, the citrate anhydride-modified polylysine is prepared by the following method: 50 mg of polylysine was dissolved in 2 mL of PBS buffer, 87.4 mg of citrate anhydride was added, the pH was adjusted to neutral with 5 mol / L sodium hydroxide solution, and the mixture was stirred overnight at room temperature. After dialyzing through a dialysis bag with a molecular weight cutoff of 500 Da for 72 h, the mixture was lyophilized to obtain citrate anhydride-modified polylysine. In one embodiment, the polymer nanocore is prepared by the following method: Polymyxin B was mixed with citrate anhydride-modified polylysine and sonicated for 30-40 seconds. The resulting complex was emulsified with polylactic acid-glycolic acid copolymer in an organic phase and then added to an ethanol solution containing 40-60% hyaluronic acid by volume. The polymer nanocore was obtained by rotary evaporation and dialysis purification.

[0027] In one embodiment, the polymer nanocore is prepared by the following method: Polymyxin B and citrate anhydride-modified polylysine were mixed at a mass ratio of 1:1 and sonicated for 30 seconds. 1 mg of the resulting complex was dissolved in 100 μL of ultrapure water, and 5 mg of polylactic acid-glycolic acid copolymer was dissolved in 1 mL of dichloromethane. The mixture was then sonicated for 1 minute. The resulting emulsion was added to 25 mL of 50% ethanol solution containing 5 mg of hyaluronic acid, stirred at room temperature for 10 minutes, and the organic solvent was removed by rotary evaporation at 30 °C. Dialysis was then performed for 72 hours using a dialysis bag with a molecular weight cutoff of 3500 Da.

[0028] The third aspect of this invention provides the application of the above-mentioned biomimetic polymyxin B nanoparticles in the preparation of a drug for treating Pseudomonas aeruginosa lung infection.

[0029] The nanoparticles provided by this invention can reduce the bacterial load in the lungs of mice infected with Pseudomonas aeruginosa, improve inflammatory factors such as TNF-α, IL-6, and IL-1β, as well as blood routine indicators such as white blood cells, granulocytes, and platelets, and reduce lung tissue damage, indicating that they can be used to prepare drugs for treating Pseudomonas aeruginosa lung infections.

[0030] Beneficial effects 1. This invention provides a biomimetic polymyxin B-loaded nanoparticle, which can be used to prepare drugs for treating Pseudomonas aeruginosa lung infections and can reduce the cytotoxicity of free polymyxin B, thus balancing antibacterial efficacy and drug safety, and better meeting the needs of practical applications.

[0031] 2. This invention employs a polymer nanocore loaded with polymyxin B and citrate anhydride-modified polylysine. By utilizing the charge reversal property of citrate anhydride-modified polylysine in an acidic environment, the nanoparticles expand in the acidic environment of infection, promoting the release of polymyxin B, which is beneficial to improving drug exposure and bactericidal effect at the infection site.

[0032] 3. This invention coats the polymer nanocore with a biomimetic layer of the macrophage membrane. The biomimetic layer can retain macrophage membrane characteristic proteins such as CD11b and CCR2, which is beneficial to improving the biocompatibility of nanoparticles and reducing the cytotoxicity of free polymyxin B.

[0033] 4. This invention loads an anti-biofilm cyclic peptide onto the surface of a biomimetic layer of a macrophage membrane. This cyclic peptide can inhibit the activity of Pseudomonas aeruginosa aminopeptidase PaAP, thereby interfering with the biofilm of Pseudomonas aeruginosa. Together with the bactericidal effect of polymyxin B, it enhances the antibacterial effect against Pseudomonas aeruginosa.

[0034] 5. The nanoparticles provided by this invention can reduce the bacterial load in the lungs of mice infected with Pseudomonas aeruginosa, improve inflammatory factors such as TNF-α, IL-6, and IL-1β, as well as blood routine indicators such as white blood cells, granulocytes, and platelets, and reduce lung tissue damage, indicating that they can be used to prepare drugs for treating Pseudomonas aeruginosa lung infections. Attached Figure Description

[0035] Figure 1 The results show the Zeta potential and particle size distribution of different groups of nanoparticles. A represents the Zeta potential of PLGA / PC-PMB nanocores, MM, and MM-EK@PLGA / PC-PMB nanoparticles at pH 7.4 and pH 5.5, while B represents the particle size distribution of PLGA / PC-PMB nanocores and MM-EK@PLGA / PC-PMB nanoparticles at pH 7.4 and pH 5.5. Figure 2The images show the transmission electron microscopy results of different groups of nanoparticles, where A represents the PLGA / PC-PMB nanocore, B represents the MM-EK@PLGA / PC-PMB nanoparticles under pH 7.4 conditions, and C represents the MM-EK@PLGA / PC-PMB nanoparticles under pH 5.5 conditions. Figure 3 Results of macrophage membrane coating and membrane protein retention are shown in Figure A, where SDS-PAGE results of PMB, MM, and MM@PLGA / PC-PMB are presented, and Western blot results of CD11b, CCR2, and Na are presented. + / K + -Results of ATPase; Figure 4 The results show the stability and in vitro release of the nanoparticles. A represents the changes in nanoparticle size and PDI at 4°C, and B represents the cumulative release of polymyxin B at pH 7.4 and pH 5.5. Figure 5 The results show the effects of different concentrations of free polymyxin B, blank carrier MM-EK@PLGA / PC, and MM-EK@PLGA / PC-PMB nanoparticles on the survival rate of RAW264.7 cells; Figure 6 The results show the minimum inhibitory concentration determination results for different formulations, where A is free polymyxin B, B is the blank carrier MM-EK@PLGA / PC, and C is MM-EK@PLGA / PC-PMB nanoparticles. Figure 7 The in vitro antibacterial colony plots for different groups; Figure 8 The results of live / dead staining of Pseudomonas aeruginosa after different treatment groups; Figure 9 Crystal violet staining results showing the effects of different groups on the established Pseudomonas aeruginosa biofilm; Figure 10 The results of detecting inflammatory factors in mouse lung tissue homogenates after different treatment groups are shown from left to right: TNF-α, IL-6, IL-1β and IL-10. Figure 11 The results of white blood cell, granulocyte, and platelet counts in mice after treatment in different groups; Figure 12 H&E staining results of lung tissue from mice after treatment in different groups; Figure 13 Results of bacterial load in the lungs of mice after treatment using bronchoalveolar lavage fluid from different groups. Detailed Implementation

[0036] The following examples are used to further illustrate the technical solutions of this application and should not be construed as limiting the scope of protection of this application. Unless otherwise specified, the methods used in the following examples are conventional; the materials and reagents used are commercially available unless otherwise specified; the room temperature is 25°C; and the pH of the PBS buffer is 7.4.

[0037] For ease of description, in the following embodiments and figures: PMB represents polymyxin B, PC represents citrate anhydride-modified polylysine, MM represents macrophage membrane nanoparticles, MM-EK represents cyclic peptide-modified macrophage membrane nanoparticles, PLGA / PC-PMB represents polymer nanocore loaded with polymyxin B, and MM-EK@PLGA / PC-PMB represents biomimetic polymyxin B-loaded nanoparticles. Figure 3 MM@PLGA / PC-PMB represents macrophage membrane-coated nanoparticles without cyclic peptide loading. MM-EK@PLGA / PC represents macrophage membrane-coated blank carriers without polymyxin B-modified cyclic peptides.

[0038] Example 1 This embodiment provides a biomimetic polymyxin B-loaded nanoparticle, comprising a polymer nanocore and a macrophage membrane biomimetic layer coating the surface of the polymer nanocore; the polymer nanocore is loaded with polymyxin B and citrate anhydride-modified polylysine; and the surface of the macrophage membrane biomimetic layer is loaded with an anti-biomembrane cyclic peptide.

[0039] The amino acid sequence of the anti-biomembrane cyclic peptide is ERWGHDFIK.

[0040] In the anti-biomembrane cyclic peptide, the carboxyl group on the side chain of glutamic acid residue and the amino group on the side chain of lysine residue are linked by isopeptide bonds to form a cyclic structure, while retaining the N-terminal amino group and C-terminal carboxyl group of the peptide chain.

[0041] The polymer nanocore also includes a polylactic acid-glycolic acid copolymer, and the preparation method of the polylactic acid-glycolic acid copolymer includes the following steps: D,L-lactide and glycolide were mixed in a mass ratio of 1:1, and 0.02% stannous octoate and 0.01% lauryl alcohol were added according to the total mass of the monomers (total mass of D,L-lactide and glycolide). The mixture was reacted at 170 °C for 2 h under vacuum. After the reaction was completed, the mixture was cooled, dissolved in anhydrous dichloromethane, and precipitated in anhydrous methanol. The precipitate was dried under vacuum at 60 °C for 48 h to obtain polylactic acid-glycolic acid copolymer.

[0042] The biomimetic polymyxin B-loaded nanoparticles have an average particle size of 172.1 nm at pH 7.4 and an average particle size of 262.1 nm at pH 5.5.

[0043] The biomimetic layer of the macrophage membrane is derived from the RAW264.7 macrophage membrane and retains the CD11b and CCR2 membrane proteins.

[0044] This embodiment also provides a method for preparing biomimetic polymyxin B-loaded nanoparticles, including the following steps: Collect 2×10 7 ~5×10 7 RAW264.7 macrophages were washed with PBS buffer and centrifuged to collect the cell pellet. The cell pellet was resuspended in ice-cold PBS buffer and centrifuged at 4°C, 600×g for 5 min. The supernatant was discarded, and the pellet was centrifuged again at 4°C, 600×g for 1 min. Membrane protein extraction reagent A containing benzyl sulfonyl fluoride was added to the resulting cell pellet, and the pellet was thoroughly resuspended. The pellet was then incubated on ice for 10–15 min, and the cells were frozen in liquid nitrogen and thawed at room temperature. This freeze-thaw cycle was repeated three times. The pellet was then centrifuged at 4°C, 700×g for 10 min, and the supernatant was collected. Finally, the pellet was centrifuged at 4°C, 14000×g for 30 min to collect the macrophage membrane. The macrophage membrane was resuspended in PBS buffer, and the membrane protein concentration was determined using the BCA method. The macrophage membrane was resuspended in ultrapure water and sonicated for 2.5 min to obtain macrophage membrane nanoparticles.

[0045] The anti-biomembrane cyclic peptide with the amino acid sequence ERWGHDFIK was mixed with the macrophage membrane nanoparticles obtained in Example 1 at room temperature for 30 min to obtain cyclic peptide-modified macrophage membrane nanoparticles; then the resulting solution was centrifuged at 20000×g for 20 min, the supernatant was discarded to remove the free anti-biomembrane cyclic peptide, and finally resuspended in PBS buffer to obtain MM-EK nanoparticles.

[0046] 50 mg of polylysine was dissolved in 2 mL of PBS buffer, and 87.4 mg of citrate anhydride was added. The pH was then adjusted to neutral with 5 mol / L sodium hydroxide solution. After stirring overnight at room temperature, the reaction solution was added to a dialysis bag with a molecular weight cutoff of 500 Da and dialyzed for 72 h for purification. The solution was then lyophilized to obtain citrate anhydride-modified polylysine, denoted as PC. The zeta potential of the PC solution was negative.

[0047] Polymyxin B was mixed with the previously prepared PC at a mass ratio of 1:1 and sonicated for 30 s to obtain a PC-PMB complex. 1 mg of the PC-PMB complex was dissolved in 100 μL of ultrapure water, and 5 mg of polylactic acid-glycolic acid copolymer was dissolved in 1 mL of dichloromethane. Both solutions were vortexed until fully dissolved. The two solutions were mixed and sonicated for 1 min using an ultrasonic cell disruptor to obtain an emulsion. 5 mg of hyaluronic acid was dissolved in 25 mL of 50% ethanol solution. The resulting emulsion was added to the ethanol solution, and the mixture was magnetically stirred at room temperature for 10 min, followed by rotary evaporation at 30 °C to remove the organic solvent. The resulting dispersion was dialyzed for 72 h in a dialysis bag with a molecular weight cutoff of 3500 Da to remove free polymyxin B, yielding a PLGA / PC-PMB nanocore.

[0048] The aforementioned MM-EK nanoparticles were mixed with the aforementioned PLGA / PC-PMB nanocores, wherein the mass ratio of macrophage membrane to polymyxin B was 1:1. The mixture was sonicated for 2.5 min to obtain biomimetic polymyxin B-loaded nanoparticles, denoted as MM-EK@PLGA / PC-PMB.

[0049] Comparative Example 1 This comparative example is free polymyxin B.

[0050] Comparative Example 2 This comparative example uses a blank carrier. The preparation method of the blank carrier includes the following steps: MM-EK nanoparticles and PC were prepared according to the method provided in Example 1. 0.5 mg of PC was dissolved in 100 μL of ultrapure water, and 5 mg of polylactic acid-glycolic acid copolymer was dissolved in 1 mL of dichloromethane. Both solutions were vortexed until fully dissolved. The two solutions were mixed and sonicated for 1 min using an ultrasonic cell disruptor to obtain an emulsion. 5 mg of hyaluronic acid was dissolved in 25 mL of 50% ethanol solution. The resulting emulsion was added to the ethanol solution, and the mixture was magnetically stirred at room temperature for 10 min, followed by rotary evaporation at 30°C to remove the organic solvent. The resulting dispersion was dialyzed for 72 h in a dialysis bag with a molecular weight cutoff of 3500 Da to obtain a PLGA / PC blank nanocore without polymyxin B. MM-EK nanoparticles were mixed with the obtained PLGA / PC blank nanocore to obtain a cyclic peptide-modified macrophage membrane-coated blank carrier without polymyxin B, denoted as MM-EK@PLGA / PC.

[0051] Performance testing 1. The Zeta potentials of the MM, MM-EK@PLGA / PC-PMB nanoparticles and PLGA / PC-PMB nanocores obtained in Example 1 were measured using dynamic light scattering at 25°C. The particle size distributions of the PLGA / PC-PMB nanocores and MM-EK@PLGA / PC-PMB nanoparticles were also measured. The results are as follows: Figure 1 As shown, the average particle size of the PLGA / PC-PMB nanocores was 165.27±7.66 nm, and the Zeta potential was -20.03±0.40 mV; MM exhibited negative charge. After MM-EK coating, the average particle size of MM-EK@PLGA / PC-PMB nanoparticles was 172.1±5.5 nm, and the Zeta potential was -41.5±5.3 mV under pH 7.4 conditions; under pH 5.5 conditions, the average particle size increased to 262.1±0.57 nm, and the Zeta potential changed to 8.62±0.97 mV. The changes in particle size and surface potential support the coating of polymer nanocores by macrophage membranes, and acidic conditions can further induce charge reversal and particle expansion in the nanoparticles.

[0052] 2. The morphology of different groups of nanoparticles was observed using transmission electron microscopy. The nanoparticle solution was dropped onto a copper grid and allowed to stand for 2 minutes. Excess liquid was absorbed with filter paper, and the nanoparticles were stained with 2 wt% phosphotungstic acid solution for 30 seconds, then dried. The nanoparticles were observed under an accelerating voltage of 120 kV. The results are as follows: Figure 2 As shown, both the PLGA / PC-PMB nanocore and the MM-EK@PLGA / PC-PMB nanoparticles under pH 7.4 conditions are relatively uniformly distributed and spherical. Under pH 5.5 conditions, the nanoparticle size increases and the structure becomes relatively loose.

[0053] 3. To verify macrophage membrane coating and membrane protein retention, the MM obtained in Example 1 was mixed with the PLGA / PC-PMB nanocore at a macrophage membrane to polymyxin B mass ratio of 1:1 and sonicated for 2.5 min to obtain MM@PLGA / PC-PMB. SDS-PAGE and Western blot were used for detection, and the results are as follows: Figure 3 As shown, MM and MM@PLGA / PC-PMB exhibit similar protein bands in the 13–195 kDa range, and CD11b, CCR2, and Na+ can be detected in MM@PLGA / PC-PMB. + / K + -ATPase was detected, while the above membrane protein was not detected in the PMB group, indicating that the characteristic membrane proteins of macrophages were preserved after being coated with the macrophage membrane.

[0054] 4. Set up a polymyxin B concentration gradient, measure the absorbance at 215 nm and plot a standard curve.

[0055] A linear fit was performed on absorbance A against polymyxin B concentration c, yielding a standard curve of A = 6.8886c + 0.1004, with a coefficient of determination R0. 2The value was 0.9992 (where A is absorbance, c is polymyxin B concentration, and R is the coefficient of determination). Based on the standard curve, the drug loading rate of MM-EK@PLGA / PC-PMB nanoparticles was 10.22 ± 0.05%, and the encapsulation efficiency was 80.14 ± 0.07%.

[0056] 5. The newly prepared MM-EK@PLGA / PC-PMB nanoparticles were placed at 4℃ and allowed to stand. The particle size and PDI were measured daily using dynamic light scattering method, and the results were observed continuously for 10 days. The results are as follows: Figure 4 As shown in Figure A, the particle size and PDI of the nanoparticles did not change significantly during the observation period, indicating that the nanoparticles have good storage stability.

[0057] 6. Take 2 mL of MM-EK@PLGA / PC-PMB nanoparticle dispersion and place it in a dialysis bag with a molecular weight cutoff of 3500 Da. Immerse the bag in 20 mL of PBS buffer (pH 5.5 or pH 7.4) and release the nanoparticles in vitro at 37°C and 100 rpm. Samples were taken at 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h, and an equal volume of buffer was added. The results are as follows: Figure 4 As shown in Figure B, 45.1% of polymyxin B was released cumulatively over 24 hours at pH 7.4, and 77.9% of polymyxin B was released cumulatively over 24 hours at pH 5.5, indicating that acidic conditions promote the release of polymyxin B.

[0058] 7. RAW264.7 macrophages were seeded into 96-well plates, approximately 2500 cells per well, with 5 replicates per group. The cells were pre-cultured at 37°C and 5% (v / v) CO2 for 12 h. After cell attachment, free polymyxin B or MM-EK@PLGA / PC-PMB nanoparticles were added to each well at final concentrations of 8 μg / mL, 16 μg / mL, 32 μg / mL, 64 μg / mL, 128 μg / mL, and 256 μg / mL, respectively. The blank vector obtained in Comparative Example 2 was added in equal amounts to the corresponding nanoparticle groups. After culturing for another 24 h, the original culture medium was discarded, and 10 μL of LCK-8 solution and 90 μL of complete culture medium were added to each well. After culturing for 2 h, OD450 was measured, and cell viability was calculated.

[0059] The results are as follows Figure 5 As shown, when blank carrier was added according to the carrier mass corresponding to each drug-loaded nanoparticle group, the cell survival rate of each dose group was approximately 95%. Free polymyxin B showed significant cytotoxicity at 64 μg / mL, and the cell survival rate was less than 60% at 256 μg / mL. The cell survival rate of MM-EK@PLGA / PC-PMB nanoparticles was still approximately 80% at 256 μg / mL. These results indicate that, compared with free polymyxin B, the biomimetic nanoparticles can reduce cytotoxicity.

[0060] 8. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of different formulations against *Pseudomonas aeruginosa* were determined using the micro-broth dilution method. The drug was diluted twofold with LB medium, with a concentration gradient of 0.125–128 μg / mL. *Pseudomonas aeruginosa* bacterial suspension was added and incubated at 37°C for 18 h. The MIC was determined based on OD600. For micro-incubation, bacterial suspensions from wells without turbidity were inoculated onto agar plates and incubated overnight at 37°C. The MBC was determined based on the colony count.

[0061] The results are as follows Figure 6 As shown, the minimum inhibitory concentration (MIC) of free polymyxin B was 2 μg / mL and the minimum bactericidal concentration (MBC) was 8 μg / mL; the MIC of the blank carrier MM-EK@PLGA / PC was 8 μg / mL and the MBC was greater than 128 μg / mL; the MIC of MM-EK@PLGA / PC-PMB nanoparticles was 1 μg / mL and the MBC was 4 μg / mL. These results indicate that MM-EK@PLGA / PC-PMB nanoparticles possess good antibacterial and bactericidal effects.

[0062] 9. Culture Pseudomonas aeruginosa to the logarithmic growth phase and adjust the concentration to 1×10⁻⁶. 7 CFU / mL of the bacterial culture was added to the blank vector MM-EK@PLGA / PC, free polymyxin B, and MM-EK@PLGA / PC-PMB nanoparticles, respectively, with PBS buffer as a control. The drug-containing groups were treated with the same final concentration of polymyxin B, while the blank vector groups were treated with the corresponding mass of vector as the nanoparticle groups. All samples were incubated at 37°C for 12 h. After incubation, the bacterial suspension was diluted and plated on agar plates, incubated at 37°C for 16 h, and the colony count was recorded to calculate the antibacterial efficiency.

[0063] The results are as follows Figure 7 As shown, the number of colonies treated with MM-EK@PLGA / PC-PMB nanoparticles was significantly reduced, and the antibacterial efficiency reached 97.43%, which is better than that of free polymyxin B and blank carrier.

[0064] The above-mentioned different preparations were co-incubated with Pseudomonas aeruginosa at 37°C for 8 hours. After centrifugation and washing, live / dead bacteria were stained using DMAO / PI. The results are as follows: Figure 8 As shown, the PBS group exhibited the strongest green fluorescence; the blank carrier group showed weaker red fluorescence; the free polymyxin B group showed enhanced red fluorescence; and the MM-EK@PLGA / PC-PMB nanoparticle group showed the weakest green fluorescence and the strongest red fluorescence, further demonstrating that the MM-EK@PLGA / PC-PMB nanoparticles have a good bactericidal effect.

[0065] 10. Adjust the concentration of Pseudomonas aeruginosa to 1×10⁻⁶. 6CFU / mL, 100 μL was inoculated into a 96-well plate and incubated statically at 37℃ for 3 days to form a biofilm. The supernatant was aspirated and the plate was washed three times with PBS buffer. Blank vector MM-EK@PLGA / PC, free polymyxin B, and MM-EK@PLGA / PC-PMB nanoparticles were added, with PBS buffer as a control. The plates were incubated at 37℃ for 24 h. After incubation, the plates were washed three times with PBS buffer, fixed with 200 μL of methanol for 15 min, air-dried, and then stained with 100 μL of 0.1 wt% crystal violet for 15 min. The plates were then washed with PBS buffer and air-dried again. The biofilm was observed under a microscope, and the absorbance at 590 nm was measured by dissolving the crystal violet in 95% (v / v) ethanol.

[0066] The results are as follows Figure 9 As shown, the PBS group retained the most biofilm, while the blank carrier and free polymyxin B could reduce the formed biofilm. The MM-EK@PLGA / PC-PMB nanoparticle group had the least biofilm, and its OD590 was significantly lower than that of the other groups, indicating that the MM-EK@PLGA / PC-PMB nanoparticles can effectively reduce or remove the formed Pseudomonas aeruginosa biofilm.

[0067] 11. A *Pseudomonas aeruginosa* pulmonary infection model was established. Mice were randomly divided into a blank group, a model group (PA), a blank vector MM-EK@PLGA / PC group, a free polymyxin B group, and a MM-EK@PLGA / PC-PMB nanoparticle group. Healthy mice were included as a blank group. Two hours post-infection, the first drug was administered via tail vein, and a second drug was administered four hours post-infection, with each administration volume being 30 μL. The free polymyxin B group and the MM-EK@PLGA / PC-PMB nanoparticle group received a dosage of 4 mg / kg based on polymyxin B. The blank vector group received the same amount of vector as the nanoparticle group, and the model group received an equal volume of PBS buffer. Twenty-four hours after the second drug administration, the lungs were flushed five times with cold PBS via endotracheal intubation, and bronchoalveolar lavage fluid (BALF) was collected, using a total of 2 mL of PBS. The BALF was centrifuged at 100 × g for 10 min to separate the supernatant from the cell particles. The concentrations of TNF-α, IL-1β, IL-6, and IL-10 in the culture supernatant were detected using enzyme-linked immunosorbent assay (ELISA). BALF supernatant was serially diluted and inoculated onto MH agar plates, and colony-forming unit (CFU) counts were determined. Mice were euthanized after treatment, and blood samples were collected for routine blood tests. Lung tissue was collected for H&E staining.

[0068] The results are as follows Figure 10As shown, compared with the blank group, the model group showed increased levels of TNF-α, IL-6, and IL-1β, and decreased levels of IL-10, indicating that infection induced a significant pulmonary inflammatory response. The blank carrier, free polymyxin B, and MM-EK@PLGA / PC-PMB nanoparticles all improved the above inflammatory factors to varying degrees, with the MM-EK@PLGA / PC-PMB nanoparticle group showing the greatest improvement.

[0069] The results are as follows Figure 11 As shown, compared with the model group, MM-EK@PLGA / PC-PMB nanoparticles can significantly reduce white blood cell and granulocyte counts and increase platelet counts, and their improvement effect is better than that of blank carrier and free polymyxin B.

[0070] The results are as follows Figure 12 As shown, compared with the PBS group, the alveolar structure of the model group was significantly damaged and accompanied by a large number of inflammatory cells infiltrating; the blank vector group and the free polymyxin B group could reduce lung tissue damage to varying degrees; the MM-EK@PLGA / PC-PMB nanoparticle group had significantly reduced inflammatory cell infiltration and the alveolar structure was close to that of the blank group.

[0071] The results are as follows Figure 13 As shown, the bacterial load in the BALF of the MM-EK@PLGA / PC-PMB nanoparticle group was significantly lower than that of the model group, the blank vector group, and the free polymyxin B group, indicating that the biomimetic polymyxin B-loaded nanoparticles provided in this application can effectively reduce the bacterial load in the lungs of mice infected with Pseudomonas aeruginosa.

Claims

1. A biomimetic polymyxin B-loaded nanoparticle, characterized in that, It includes a polymer nanocore and a macrophage membrane biomimetic layer coated on the surface of the polymer nanocore; the polymer nanocore is loaded with polymyxin B and citrate anhydride-modified polylysine; the surface of the macrophage membrane biomimetic layer is loaded with anti-biomembrane cyclic peptides.

2. The biomimetic polymyxin B-loaded nanoparticles according to claim 1, characterized in that, The amino acid sequence of the anti-biomembrane cyclic peptide is ERWGHDFIK; the side chain carboxyl group of the glutamic acid residue and the side chain amino group of the lysine residue in the anti-biomembrane cyclic peptide are connected by isopeptide bonds to form a cyclic structure, and the N-terminal amino group and C-terminal carboxyl group of the peptide chain are retained.

3. The biomimetic polymyxin B-loaded nanoparticles according to claim 1, characterized in that, The polymer nanocore also includes polylactic acid-glycolic acid copolymer.

4. The biomimetic polymyxin B-loaded nanoparticles according to claim 1, characterized in that, The biomimetic polymyxin B-loaded nanoparticles have a particle size of 100~300nm.

5. The biomimetic polymyxin B-loaded nanoparticles according to claim 1, characterized in that, The biomimetic layer of the macrophage membrane is derived from the RAW264.7 macrophage membrane and retains the CD11b and CCR2 membrane proteins.

6. The biomimetic polymyxin B-loaded nanoparticles according to claim 1, characterized in that, The mass ratio of polymyxin B to citrate anhydride-modified polylysine is 0.5-2:

1.

7. The biomimetic polymyxin B-loaded nanoparticles according to claim 1, characterized in that, The mass ratio of the macrophage membrane to polymyxin B is 0.5-2:

1.

8. A method for preparing biomimetic polymyxin B-loaded nanoparticles according to any one of claims 1-7, characterized in that, Includes the following steps: Macrophage cell membranes were extracted and subjected to ultrasonic treatment to obtain macrophage cell membrane nanoparticles; Anti-biomembrane cyclic peptides were mixed with macrophage membrane nanoparticles to obtain cyclic peptide-modified macrophage membrane nanoparticles. Polylysine was reacted with citrate anhydride to obtain citrate anhydride-modified polylysine. Polymyxin B was mixed with citrate anhydride-modified polylysine and then encapsulated with polylactic acid-glycolic acid copolymer to obtain a polymer nanocore loaded with polymyxin B. Cyclic peptide-modified macrophage membrane nanoparticles were mixed with the polymer nanocore and subjected to ultrasonic treatment to obtain the biomimetic polymyxin B-loaded nanoparticles.

9. The preparation method according to claim 8, characterized in that, The macrophage membrane was obtained from RAW264.7 macrophages through repeated freeze-thaw cycles and differential centrifugation. The obtained macrophage membrane was then sonicated for 2-3 minutes to obtain macrophage membrane nanoparticles.

10. The use of the biomimetic polymyxin B nanoparticles according to any one of claims 1-7 in the preparation of a drug for treating Pseudomonas aeruginosa lung infection.

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