Cell membrane modified inflammation-responsive release nanoparticles, and preparation method and application thereof

Abstract

This invention discloses a cell membrane-modified inflammatory-responsive release nanoparticle, its preparation method, and its applications. The nanoparticles use a polylactic-co-glycolic acid (PLA) core, and their surface is modified with an immune cell membrane. The PLA is loaded with an antibacterial drug and modified with a positively charged material and a responsive switch. The immune cell membrane is obtained by isolating immune cells pre-stimulated by pathogens. The nanoparticles prepared by this invention possess the function of rapidly releasing drugs targeting homologous bacteria and sites of inflammation. During systemic circulation, they tend to target sites of inflammation caused by bacterial infection, rapidly releasing the drug under the action of the highly expressed MMP-3 enzyme at the site of inflammation, achieving efficient delivery of antibacterial drugs. This invention has shown good therapeutic effects in a mouse model of bacterial infection, providing not only an effective means for the in vivo delivery of cationic antimicrobial peptides but also a promising therapeutic strategy against Gram-negative bacterial infections.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a cell membrane-modified inflammatory-responsive release nanoparticle, its preparation method, and its application. Background Technology

[0002] Multidrug-resistant bacteria (MDRs) are bacteria resistant to three or more drugs. They are highly infectious and lethal, posing a significant challenge to the medical field today. In the World Health Organization's global list of priority antibiotic-resistant bacteria, Gram-negative bacteria are ranked at the highest level. Therefore, there is an urgent need to develop safe and effective therapies to prevent and treat bacterial infections, especially those caused by Gram-negative bacteria.

[0003] Compared to traditional small-molecule antibiotics, antimicrobial peptides offer advantages such as high efficacy and selective toxicity. Their multi-mechanism and multi-target antimicrobial properties significantly reduce the likelihood of bacterial resistance. Their main mechanisms of action are direct inhibition or killing of pathogenic microorganisms and regulation of the body's immune function. Clinically, they can be used as standalone antimicrobial agents or in combination with traditional antibiotics to exert synergistic effects. Currently, colistin is considered a "last-line" drug for treating infections caused by extensively drug-resistant Gram-negative bacteria, but it suffers from drawbacks such as high nephrotoxicity and neurotoxicity, a narrow therapeutic window, and poor lung penetration. With the increasing clinical use of colistin and the spread of the resistance plasmid mcr-1, the problem of colistin-resistant strain infections is becoming increasingly serious. Furthermore, antimicrobial peptides have disadvantages in systemic circulation, including short half-life, high clearance rate, and low concentration at the site of bacterial infection, thus requiring higher dosages to improve antimicrobial efficacy, especially against drug-resistant bacteria. However, this also brings significant safety risks and higher treatment costs. Therefore, improving the stability of antimicrobial peptides in vivo and their aggregation at bacterial infection sites to achieve both high antimicrobial efficacy and good biosafety is an important solution.

[0004] Immune cells are a diverse core cell group in the human immune system, encompassing multiple subtypes such as lymphocytes, phagocytes, and dendritic cells. They are widely distributed throughout the body's tissues and organs, as well as in the peripheral blood circulation, accounting for over 99% of nucleated cells in peripheral blood. They are crucial for defending against pathogens (bacteria, viruses, fungi, etc.), clearing abnormal cells (such as cancerous and senescent cells), and precisely regulating the balance of immune responses. When the body encounters infection, injury, or inflammatory responses, immune cells are activated by various cytokines (such as TNF-α, IL-2, and IFN-γ). Their activity and functional state undergo dynamic adjustments, and guided by the concentration gradient formed by chemokines (such as CCL2, CXCL8, and CXCL10), they break through tissue barriers and are recruited to lesions or damaged sites to initiate targeted immune defense and repair processes. With the rise of cell membrane biomimetic nanocarriers, researchers have discovered that cell membrane vesicles derived from immune cells inherit unique biological characteristics from their parent cells. They not only possess excellent immunocompatibility, circumventing immune rejection, but also have natural tissue-targeting capabilities, precisely targeting inflammatory lesions or the tumor microenvironment. For example, Zhang et al. used macrophage membranes to encapsulate liposomes carrying emodin, which can recognize chemokine gradients, significantly targeting inflammatory sites in a dextran sulfate sodium (DSS)-induced mouse colitis model, achieving better therapeutic effects than free drugs. Li used T lymphocyte membranes to encapsulate nanoparticles carrying chemotherapeutic drugs, leveraging the specific recognition ability of T cells for tumor cells to achieve highly efficient drug accumulation in tumor tissues in a melanoma mouse model, significantly improving anti-tumor effects and reducing toxic side effects. However, the technical effects of existing technologies are not ideal, making it difficult to achieve precise targeting of bacteria. Summary of the Invention

[0005] Purpose of the invention: To address the problems existing in the prior art, the present invention provides a cell membrane-modified inflammatory-responsive release nanoparticle, which is a nano-drug delivery system that can efficiently target bacteria and rapidly release drugs at the bacterial infection site, thereby achieving efficient delivery of antibacterial drugs.

[0006] Another object of the present invention is to provide a method for preparing and applying the cell membrane-modified inflammatory-responsive release nanoparticles.

[0007] Technical solution: To achieve the above objective, the present invention provides a cell membrane-modified inflammatory-responsive release nanoparticle, wherein the nanoparticle has a polylactic acid-glycolic acid copolymer (PLGA) core and its surface is modified with an immune cell membrane; wherein the polylactic acid-glycolic acid copolymer is loaded with an antibacterial drug and modified with a positively charged material and a responsive switch; the immune cell membrane is a cell membrane obtained by isolating immune cells pre-stimulated by pathogens.

[0008] The antibacterial drug is an antibacterial polypeptide drug.

[0009] The positively charged material includes any one or more of polyvinyl alcohol, polysorbate, sodium dodecyl sulfate, chitosan, gelatin, and gum arabic.

[0010] The responsive switch includes any one or more of the following: MMP-2 protease substrate, MMP-3 protease substrate, MMP-9 protease substrate, elastase substrate, esterase substrate, β-lactamase substrate, neuraminidase substrate, polyhistidine, polyacrylic acid, polysulfide, polyselenoside, metal-organic framework, and anti-LPS antibody.

[0011] The immune cells include any one or more of macrophages, T lymphocytes, B lymphocytes, dendritic cells, natural killer cells, neutrophils, mast cells, eosinophils, and basophils.

[0012] The pathogens include any one or more of Escherichia coli, Salmonella, Shigella, Pseudomonas aeruginosa, Haemophilus influenzae, Klebsiella pneumoniae, Acinetobacter baumannii, and Neisseria gonorrhoeae.

[0013] The method for preparing cell membrane-modified inflammatory-responsive release nanoparticles according to the present invention includes the following steps:

[0014] (1) Synthesis of drug-loaded nanoparticles

[0015] a. Dissolve the carrier material PLGA in a solvent and stir to obtain an organic phase;

[0016] b. Dissolve the antibacterial drug and the responsive switch in water and stir to obtain an aqueous phase;

[0017] c. Add the aqueous phase to the organic phase and stir to react and obtain the primary emulsion;

[0018] d. The promulgated material is added to an external aqueous solution containing a positively charged material, stirred to react, washed, and dried to obtain nanoparticles;

[0019] (2) Preparation of cell membrane vesicles pre-stimulated by pathogens

[0020] e. Culture immune cells to the logarithmic phase;

[0021] f. Add pathogens to stimulate the body;

[0022] g. After centrifuging to collect cells, perform sonication and centrifugation, collect the supernatant and centrifuge again to obtain cell membrane vesicles;

[0023] (3) Modification of drug-loaded nanoparticles

[0024] The nanoparticles obtained in step d were dissolved in water, mixed with the cell membrane vesicles obtained in step g, and then sonicated to obtain cell membrane-modified inflammatory-responsive nanoparticles.

[0025] Preferably, the solvent in step (1)a is dichloromethane, trifluoroethanol, or Span 80, with a volume ratio of 1:0.1 to 1:0.1% to 10%.

[0026] The ratio (m / v, mg / mL) of the carrier material and the mixed solvent mentioned in step (1)a is 1:0.1~10.

[0027] The ratio of the antimicrobial drug, responsive switch, and water in step (1)b (m / m / v, mg / mg / mL) is 1:0.05~1:0.01~2.

[0028] The volume ratio of the colostrum and the external aqueous phase in step (1)d is 1:20~200.

[0029] The MOI of the pathogen mentioned in step (2)f is 1~20.

[0030] In step (3), the mass ratio of nanoparticles to vesicles (based on total protein content) is 1:0.1~5.

[0031] Further, the stirring temperature in step (1)a is 0~50℃; the stirring temperature in step (1)b is 0~50℃; the reaction temperature in step (1)c is 0~50℃; the reaction time in step (1)c is 1~180 min; the reaction temperature in step (1)d is 0~50℃; and the reaction time in step (1)d is 0.1~5 h.

[0032] The stimulation time described in step (2)f is 0.5-7 days.

[0033] The processing temperature in step (3) is 0~37℃; the processing time in step (3) is 1~60 min;

[0034] The ultrasonic power mentioned in step (3) is 10-1000W; the nanoparticles mentioned in step (3) are stored at 0~20℃.

[0035] Preferably, the cell membrane-modified inflammatory-responsive release nanoparticles are PLGA nanoparticles, and the surface of the nanoparticles is modified with immune cell membrane modification.

[0036] The cell membrane is a cell membrane obtained by separating immune cells after pre-stimulation by pathogens;

[0037] The PLGA nanoparticles are loaded with antibacterial drugs and modified with NFF-3 peptide and chitosan.

[0038] Furthermore, the immune cells are macrophages.

[0039] Furthermore, the antibacterial drug is an antimicrobial peptide drug.

[0040] Furthermore, the cell membrane-modified inflammatory-responsive release nanoparticles have a particle size of 200-220 nm and a zeta potential of -3 to -6 mV.

[0041] Preferably, the method for preparing the above-mentioned cell membrane-modified inflammatory-responsive release nanoparticles includes the following steps:

[0042] S1. Extract immune cell membrane vesicles pre-stimulated by pathogens;

[0043] S2. Prepare PLGA nanoparticles containing antibacterial drugs and modify them with NFF-3 peptide and chitosan;

[0044] S3. The nanoparticles and cell membrane are mixed and then subjected to ultrasonic treatment to obtain the product.

[0045] Further, in step S1, the method for prestimulating immune cells with pathogens includes the following steps:

[0046] Mouse mononuclear macrophage leukemia cells were cultured in a medium containing fetal bovine serum and then stimulated with bacteria (MOI=10) for 36 hours.

[0047] Further, in step S1, the method for extracting the immune cell membrane vesicles includes the following steps:

[0048] Mouse mononuclear macrophage leukemia cells pre-stimulated with pathogens were cultured in a medium containing fetal bovine serum for 36 hours. After centrifugation, the cells were collected and then subjected to sonication and centrifugation. The supernatant was collected and centrifuged again to obtain cell membrane vesicles.

[0049] Further, in step S2, the preparation method of the PLGA nanoparticles includes the following steps:

[0050] PLGA was dissolved in a solvent and stirred in an ice bath to obtain an organic phase; an antibacterial drug and NFF-3 peptide were dissolved in a solvent and stirred to obtain an aqueous phase; the aqueous phase was added to the organic phase and stirred in an ice bath to obtain a promulgation; the promulgation was added to a polyvinyl alcohol (PVA) solution and stirred in an ice bath to solidify; the nanoparticles were added to a chitosan solution, stirred, centrifuged, and washed to obtain the PLGA nanoparticles.

[0051] The application of the cell membrane-modified inflammatory-responsive release nanoparticles described in this invention in the preparation of antibacterial drugs.

[0052] The application of the cell membrane-modified inflammatory-responsive release nanoparticles in the preparation of drugs for treating bacterial infections and related diseases caused by bacterial infections.

[0053] The application of the cell membrane-modified inflammatory-responsive release nanoparticles described in this invention in the in vivo delivery of cationic peptide drugs.

[0054] This invention first synthesizes NFF-3 peptide-modified polylactic-co-glycolic acid (PLGA) nanoparticles loaded with the antimicrobial peptide S-Thanatin (Ts) via a double emulsion method (Ts@PN). Then, chitosan (CS) is applied to the surface of the PLGA nanoparticles (Ts@CPN). Next, macrophage membrane vesicles (PMs) pre-stimulated by pathogens are extracted. Finally, the PMs are coated onto the surface of the Ts@CPN nanoparticles to obtain cell membrane-modified inflammatory-responsive release nanoparticles (Ts@CPN@PM). The Ts@CPN@PM nanoparticles prepared in this invention possess the function of PM targeting bacteria and responsively degrading NFF-3 peptides in a high MMP-3 enzyme environment. During systemic circulation, they initially tend to target the inflammatory sites caused by bacterial infection, and are then further degraded by the highly expressed MMP-3 enzyme at the inflammatory site, achieving efficient delivery and rapid release of the antimicrobial peptide. Ts@CPN@PM showed good therapeutic effects in a mouse model of bacterial infection, becoming a promising strategy against Gram-negative bacterial infection.

[0055] The cell membrane-modified inflammatory-responsive release nanoparticles prepared in this invention aim to solve the following problems:

[0056] 1. Immunocellular membrane-modified nanoparticles exhibit weak targeting of pathogens and lack infection specificity: Existing immunocellular membrane-modified nanoparticles largely rely on the natural inflammatory chemotaxis or non-specific recognition of immune cells, making it difficult to achieve precise identification of specific pathogens and enrichment in deep infection lesions. For example, ordinary macrophage membrane-modified nanoparticles (such as CN118320115A and CN116407615A) mainly rely on natural TLR recognition or inflammatory chemotaxis, lacking specific binding to pathogens; NK cell membrane-modified nanoparticles (such as CN120531700A), although capable of targeting inflammation, lack direct binding ability to pathogens, making it difficult to precisely accumulate in intracellular bacteria or deep infection lesions.

[0057] 2. Traditional responsive nanoparticles often lack precise drug release triggering, leading to premature leakage or delayed release: Existing responsive drug-loaded nanoparticles frequently employ pH, ROS, and other response mechanisms, which are easily affected by fluctuations in the normal tissue microenvironment or oxidative stress, resulting in premature drug leakage or delayed release at the lesion site. For example, acid-responsive nanoparticles (CN111269367A, CN113476425B, CN115504910B, CN107714707A) are affected by physiological pH fluctuations; ROS-responsive nanoparticles (CN113577299A, CN115671298B, CN118662479A) are easily affected by oxidative stress.

[0058] 3. Insufficient synergy between antibacterial and inflammatory regulation leads to frequent recurrence of infections: During infection, bacterial proliferation and inflammatory response mutually promote each other. Relying solely on bactericidal action without synchronous regulation of the inflammatory microenvironment easily causes tissue damage and recurrence. Existing nano-formulations (such as CN118662479A) have certain bacterial targeting capabilities, but their anti-inflammatory function is insufficient to simultaneously alleviate excessive release of inflammatory factors and post-infection tissue damage.

[0059] This invention constructs a drug delivery system based on "bionic targeting, precise controlled release, and synergistic antibacterial and anti-inflammatory effects," with the core design as follows:

[0060] 1. Pathogen pre-stimulation of macrophage membrane biomimetic modification achieves infection-specific targeting and improved circulation stability.

[0061] By prestimulating macrophages with pathogens, the cell membrane surface of macrophages is enriched with specific pattern recognition receptors (such as TLR2 / 4, NOD-like receptors, etc.) for the pathogens, thereby endowing nanoparticles with the ability to actively recognize and enrich specific pathogens and infection foci. At the same time, CD47 and other "don't eat me" signaling molecules and related chemotactic / immune escape molecules are retained to reduce clearance by the mononuclear-phagocytic system, prolong blood circulation time, improve biocompatibility, and reduce immunogenicity.

[0062] 2. Inflammatory enzyme-responsive structural switch, enabling site-specific triggering of drug release.

[0063] By using a specific substrate of the MMP-3 enzyme, which is highly expressed at the site of inflammation, as a switch for the nanoparticle structure and integrating it into the carrier, the nanoparticles maintain structural stability in a normal tissue environment and reduce the risk of premature leakage. When the nanoparticles reach the site of infection and inflammation, the highly active MMP-3 enzyme triggers the switch to degrade, thereby causing the nanoparticles to disintegrate and rapidly release the loaded antibacterial drug. This achieves precise spatiotemporal release control, increases the effective drug concentration at the lesion site, and reduces systemic toxic side effects.

[0064] 3. Synergistic antibacterial and anti-inflammatory effects to block the vicious cycle of infection and inflammation.

[0065] The antibacterial drugs released by the carrier are used to directly kill bacteria and eliminate the source of infection. At the same time, the pre-stimulated macrophage membrane can neutralize excess inflammatory factors (such as TNF-α, IL-1β, IL-6, etc.) at the site of infection through inflammatory factor receptors on its surface, and participate in regulating the inflammatory response and macrophage polarization, thereby reducing tissue damage caused by inflammatory storm and achieving synergistic treatment of bactericidal and anti-inflammatory effects.

[0066] In this invention, the key parameters include pre-stimulation time, multiple of infection (MOI), the mass ratio of macrophage membrane vesicles to nanoparticles, and nanoparticle size and potential.

[0067] 1. Pre-stimulation time affects the expression level of pathogen-specific receptors: if the time is too short, the receptor induction is insufficient and the specificity of targeting is weak; if the time is too long, it is easy to lead to excessive activation and apoptosis of macrophages, and a decline in cell membrane integrity and function.

[0068] 2. The multiple of infection (MOI) determines the efficiency of pre-stimulation: too low an MOI is insufficient to effectively activate receptor pathways; too high an MOI can easily lead to excessive activation or even lysis and death of macrophages.

[0069] 3. The ratio of membrane vesicles to nanoparticles affects the modification effect and functional integrity: if the ratio is too low, the modification is insufficient and the targeting ability is weak; if the ratio is too high, it is easy to aggregate, increase the particle size, reduce tissue penetration and increase the risk of systemic toxic side effects.

[0070] 4. Particle size affects circulation time and uptake efficiency: Controlling the particle size to about 200 nm is beneficial for both circulation in the body and enrichment at the site of inflammation; if the particle size is too large, it is easily cleared by the mononuclear phagocytic system, and if it is too small, it is not conducive to drug loading and is prone to leakage.

[0071] 5. Potential affects stability and safety: Nanoparticles carry a weak negative charge, which helps reduce the toxicity of positively charged nanoparticles to normal cells and reduces non-specific adsorption with positively charged proteins, thereby reducing the probability of being cleared by the immune system.

[0072] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0073] 1. Infection-specific targeted enrichment: By pre-stimulating macrophage membranes with pathogens, the carrier is endowed with the ability to actively recognize specific pathogens, thereby improving the enrichment efficiency at the infection site and reducing exposure to non-target tissues.

[0074] 2. Site-specific drug release: The inflammatory enzyme-responsive switch triggers drug release in the inflammatory microenvironment of infection, reducing the risk of premature leakage and increasing the effective drug concentration at the lesion site, thereby improving efficacy and reducing systemic toxicity.

[0075] 3. Synergistic antibacterial and anti-inflammatory treatment: While clearing the source of bacterial infection, membrane surface receptors neutralize excess inflammatory factors and regulate the inflammatory response, which helps to reduce tissue damage and lower the risk of recurrence. It is suitable for deep infections, complicated infections and other scenarios.

[0076] 4. Advantages for delivery of peptide antibacterial drugs: Encapsulation with nanocarriers can reduce the risk of peptide antibacterial drugs being bound by plasma proteins or degraded by proteases in the bloodstream, and improve their ability to reach the site of infection, providing a feasible technical route for in vivo delivery of peptide drugs. Attached Figure Description

[0077] Figure 1 A schematic diagram of the preparation process of cell membrane-modified inflammatory-responsive release nanoparticles is presented.

[0078] Figure 2 The characterization results of cell membrane-modified inflammatory-responsive release nanoparticles are presented; among them, Figure 2 A presents the particle size distribution and transmission electron microscopy images of the nanoparticles; Figure 2 B presents the polydispersity index (PDI) of the nanoparticles. Figure 2 C shows the zeta potential of the nanoparticles; Figure 2 D presented polypropylene gel electrophoresis (SDS-PAGE) analysis of protein bands of Ts@CPN, Ts@CPN@PM and cell membrane vesicles; Figure 2 E presents drug release curves in the presence or absence of MMP-3 protease and under different pH conditions; Figure 2 F shows the particle size change of the nanoparticles over 7 days under storage conditions at 4°C.

[0079] Figure 3 The results of the in vitro antibacterial activity test were presented; among them, Figure 3 A represents the minimum inhibitory concentration (MIC) of polymyxin (PMB), free drug Ts, and two drug-loaded nanoparticles (Ts@CPN and Ts@CPN@PM) against Escherichia coli (E. coli). Figure 3 B and C are (B) bacterial plating images and (C) colony count results of E. coli after incubation of PMB, free drug Ts, and two drug-loaded nanoparticles (Ts@CPN and Ts@CPN@PM); Figure 3 D and E are (D) live / dead staining confocal microscopy images and (E) mortality rate of bacteria after co-incubation of PMB, free drug Ts, and two drug-loaded nanoparticles (Ts@CPN and Ts@CPN@PM) with E. coli.

[0080] Figure 4The results of in vitro targeting ability tests are presented; confocal microscopy images and corresponding fluorescence intensity distribution maps of two fluorescent dye nanoparticles (Cou 6@CPN and Cou 6@CPN@PM) co-incubated with E. coli are presented.

[0081] Figure 5 The results of in vivo targeting ability testing were presented; among them, Figure 5 A and B show the fluorescence imaging images and quantitative results of ex vivo tissues from unmodeled mice 2 h, 12 h, 24 h, and 36 h after tail vein injection of free fluorescent dye IR783 and two fluorescent dye-loaded nanoparticles (IR783@CPN and IR783@CPN@PM). Figure 5 C and D show the fluorescence imaging images (C) and quantitative results (D) of the ex vivo tissues of model mice 2 h, 12 h, 24 h, and 36 h after tail vein injection of free fluorescent dye IR783 and two fluorescent dye-loaded nanoparticles (IR783@CPN and IR783@CPN@PM).

[0082] Figure 6 The results of in vivo therapeutic performance tests were presented; among them, Figure 6 A is a diagram of the in vivo treatment plan; Figure 6 B represents the survival curve results; Figure 6 C represents the result of weight change; Figure 6 D and E are the lung tissue smear images (D) and colony count results (E); Figure 6 F represents the protein level detection result in bronchoalveolar lavage fluid (BALF); Figure 6 GI represents the results of the detection of (G) tumor necrosis factor α (TNF-α), (H) interleukin 1β (IL-1β) and (I) interleukin 6 (IL-6) levels in BALF; Figure 6 J represents the results of hematoxylin-eosin (H&E) staining of lung tissue.

[0083] Figure 7 The results of in vitro safety testing were presented; among them, Figure 7 A represents the hemolytic toxicity of PMB, free drug Ts, and two drug-loaded nanoparticles (Ts@CPN and Ts@CPN@PM) on blood cells at different drug concentrations; Figure 7 B represents the toxicity test results of PMB, free drug Ts, and two drug-loaded nanoparticles (Ts@CPN and Ts@CPN@PM) on A549, BEAS 2B, and RAW264.7 cells at different drug concentrations; Figure 7CE represents live / dead staining confocal microscopy images of PMB, free drug Ts, and two drug-loaded nanoparticles (Ts@CPN and Ts@CPN@PM) after co-incubation with (C) A549, (D) BEAS 2B, and (E) RAW 264.7 cells.

[0084] Figure 8 The results of in vivo safety tests were presented; among them, Figure 8 A shows the changes in body weight of mice in each group after 7 days of continuous injection of PBS, PMB, Ts, Ts@CPN and Ts@CPN@PM; Figure 8 BD represents the routine blood parameters of mice in each group after continuous injection of PBS, PMB, Ts, Ts@CPN and Ts@CPN@PM for 7 days: (B) red blood cells, (C) white blood cells and (D) platelets; Figure 8 EJ represents the blood biochemical parameters of mice in each group after continuous injection of PBS, PMB, Ts, Ts@CPN and Ts@CPN@PM for 7 days: (E) alanine aminotransferase (ALT), (F) aspartate aminotransferase (AST), (G) albumin (ALB), (H) blood urea nitrogen (BUN), (I) creatinine (CRE) and (J) uric acid (UA). Figure 8 K represents the H&E staining results of the major organs (heart, liver, spleen, lung, and kidney) of mice in each group 7 days after continuous injection of PBS, PMB, Ts, Ts@CPN, and Ts@CPN@PM. Detailed Implementation

[0085] The technical solution of the present invention will be further described below with reference to the accompanying drawings, but it is not limited thereto. Any modifications or equivalent substitutions to the technical solution of the present invention that do not depart from the spirit and scope of the technical solution of the present invention should be covered within the protection scope of the present invention.

[0086] Unless otherwise specified, all materials and reagents used in the embodiments are commercially available.

[0087] The polylactic acid-glycolic acid copolymer (PLGA) used in this embodiment of the invention was purchased from Guangzhou Weihua Biotechnology Co., Ltd. (item number: WH008N06).

[0088] The antimicrobial peptide S-Thanatin (sequence: GSKKPVPIIYCNRRSGKCQRM) in this embodiment of the invention was synthesized by Nanjing Genscript Biotech Co., Ltd.

[0089] The NFF-3 (sequence: Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg-Lys(DNP)-NH2) in this embodiment of the invention was synthesized by Nanjing Genscript Biotech Co., Ltd.

[0090] The polyvinyl alcohol 1750±50 (average molecular weight 77087.5) in the embodiments of the present invention was purchased from Sinopharm Chemical Reagent Co., Ltd. (product number: 30153160).

[0091] The chitosan (MW = 30000) in the embodiments of the present invention was purchased from Shanghai Macklin Biochemical Co., Ltd. (product number: C850346).

[0092] The Escherichia coli strain number used in the embodiments of the present invention is ATCC 25922, and the source is American Type Culture Collection (ATCC, USA).

[0093] The RAW 264.7 cell line used in the embodiments of the present invention was purchased from the Cell Bank of the Chinese Academy of Sciences.

[0094] In the embodiments of the present invention, BALB / c mice (6 - 8 weeks old, male, body weight 18 - 22 g, SPF level) were used as the experimental animal subjects, purchased from Hangzhou Ziyuan Experimental Animal Technology Co., Ltd., animal production license number: SCXK(Zhe)2024 - 0004, animal certificate number: 20250901Abzz01050000123. During the experiment, the mice could drink water freely and were raised in the clean area.

[0095] Example 1

[0096] An inflammation-responsive release nanoparticle modified with cell membrane, the nanoparticle is a core of poly(lactic-co-glycolic acid) (PLGA), and its surface is modified with immune cell membrane; wherein, the cell membrane is the cell membrane isolated from immune cells after pre-stimulation with pathogenic bacteria; the PLGA nanoparticle is loaded with antibacterial peptide S-Thanatin, and is simultaneously modified with chitosan and NFF-3 peptide (a specific substrate of MMP-3 enzyme).

[0097] The preparation method of the above-mentioned inflammation-responsive release nanoparticle modified with cell membrane ( Figure 1 shows a schematic diagram of the preparation process of the inflammation-responsive release nanoparticle modified with cell membrane), including the following steps:

[0098] S1. Pre-stimulate immune cells with pathogenic bacteria: When mouse monocyte macrophage leukemia cells (RAW 264.7) are cultured in DMEM medium containing 10% fetal bovine serum (FBS) until 60 - 70% confluent, add Escherichia coli in the logarithmic phase (MOI = 10) for stimulation, the stimulation time is 36 hours, and the temperature is 37°C;

[0099] Cell membrane vesicles of immune cells were isolated: Pre-stimulated macrophages were collected by low-speed centrifugation (800×g, 5 min) and washed with phosphate-buffered saline (PBS) (pH 7.4). Cells were dispersed in separation buffer (containing 15 mM Tris-HCl (pH 7.5), 100 mM sucrose, 2 mM magnesium chloride, 10 mM potassium chloride, and 1% protease inhibitor (BioSharp, catalog number: BL612A)). Cells were disrupted using an ultrasonic cell disruptor under ice bath conditions. The disrupted solution was centrifuged at 4°C (10000×g, 20 min) to remove cell pellet, and the supernatant was collected. The supernatant was centrifuged again (100000×g, 1 h) to collect cell membrane vesicle pellet, and finally dispersed in 1 mL PBS and stored at -80°C.

[0100] S2. Dissolve 10 mg PLGA in 2.5 mL of organic solvent (containing 2 mL dichloromethane, 500 μL trifluoroethanol, and 1% Span 80), and stir in an ice bath to obtain the organic phase. Dissolve 10 mg of the antimicrobial peptide S-Thanatin (Ts) and 1 mg of NFF-3 peptide in 500 μL of water, and stir to obtain the aqueous phase. Quickly inject the aqueous phase into the organic phase and stir in an ice bath for 5 min to obtain the proemulsion. Prepare a 2% polyvinyl alcohol (PVA) aqueous solution (2 g PVA dissolved in 100 mL of water), stir at 90 °C until dissolved, and cool to room temperature. Quickly inject the proemulsion into 100 mL of the 2% PVA aqueous solution, stir in an ice bath for 3 h to solidify, centrifuge the solidified reaction solution, remove the supernatant, and obtain the precipitate as drug-loaded PLGA nanoparticles (Ts@PN). Prepare a 0.2% chitosan (CS) aqueous solution (20 mg CS dissolved in 10 mL of 2% acetic acid aqueous solution), and stir to dissolve at 37 °C. The nanoparticles were added to 10 mL of 0.2% CS aqueous solution and stirred at room temperature for 30 min. After washing and centrifugation (8000 rpm, 30 min), the precipitate was the CS-modified nanoparticles (Ts@CPN).

[0101] The protein content of cell membrane vesicles in S3 and S1 was determined by the BCA method. Based on the total protein and the mass of antimicrobial peptides in the CS-modified nanoparticles, the vesicles were mixed at a 1:1 mass ratio and sonicated in an ice bath for 10 min (65W, 2 s on / 2 s off) to obtain Ts@CPN@PM, which is the cell membrane-modified inflammatory-responsive release nanoparticle. Depending on the requirements, it can be freeze-dried at low temperature for 24 h to obtain Ts@CPN@PM lyophilized powder, which can then be redispersed in ultrapure water before use.

[0102] The materials prepared in this embodiment Figure 2 The characterization results of cell membrane-modified inflammatory-responsive release nanoparticles are presented; among them, Figure 2A shows the particle size distribution and transmission electron microscopy images of the nanoparticles (arrows indicate that Ts@CPN@PM is modified with cell membrane vesicles); Figure 2 B presents the polydispersity index (PDI) of the nanoparticles (n=3); Figure 2 C shows the zeta potential of the nanoparticles (n=3); Figure 2 D presents polypropylene gel electrophoresis (SDS-PAGE) analysis of protein bands in Ts@CPN, Ts@CPN@PM, and pre-activated macrophage membrane-derived nanovesicles (PMNVs). The nanoparticles were stored at 4°C, and samples were taken daily to determine particle size and PDI for 7 consecutive days. Figure 2 E shows the particle size change of nanoparticles over 7 days under storage conditions at 4°C (n=3). Drug release was determined by dialysis, with 4 experimental groups: pH=7.4, no MMP-3 group; pH=7.4, containing MMP-3 group; pH=5.5, no MMP-3 group; and pH=5.5, containing MMP-3 group. Two mg of cell membrane-modified inflammatory-responsive release nanoparticles were dissolved in 2 mL of simulated lung fluid (composition (g / L): MgCl2 0.095, NaCl 6.019, KCl 0.298, Na2HPO4 0.126, Na2SO4 0.063, CaCl2·2H2O 0.368, CH3COONa 0.547, NaHCO3 2.604, Na3C6H5O7·2H2O 0.097). The solution was transferred to a dialysis bag (3.5 kDa), which was then immersed in a centrifuge tube containing 48 mL of simulated lung fluid. The mixture was incubated at 37°C and 100 rpm. Samples were taken at 0, 0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12, 18, and 24 h. Two mL of the release solution was aspirated and immediately replaced with 2 mL of fresh simulated lung fluid. The drug concentration in the release solution was determined by the BCA method. Figure 2 F presents drug release curves (n=3) with and without MMP-3 protease and under different pH conditions.

[0103] according to Figure 2 A shows that, according to transmission electron microscopy images, Ts@CPN@PM successfully modified cell membrane vesicles with a particle size of approximately 220 nm, compared to Ts@CPN. Figure 2As shown in Figure B, the polydispersity index of the nanoparticles is less than 0.20, indicating that the nanoparticles have uniform size and are evenly dispersed. In the dynamic light scattering experiment, the zata potentials of Ts@CPN, Ts@CPN@PM, and PM NVs are 7 mV, -4 mV, and -18 mV, respectively, indicating that after modifying the negatively charged cell membrane vesicles, the overall charge of Ts@CPN@PM changes from the positive charge of Ts@CPN to a negative charge. Figure 2 C). In polyacrylamide gel electrophoresis, most of the protein bands of PM were observed on Ts@CPN@PM, indicating that the membrane proteins inherited from PM were effectively retained on the surface of Ts@CPN@PM. Figure 2 D). Ts@CPN@PM stored at 4℃ for 7 days did not show significant changes in particle size and PDI. Figure 2 E) indicates that the nanoparticles have good stability under these storage conditions. In a weakly acidic environment (pH 5.5) in the presence of the MMP-3 protease, the NFF-3 peptide in the nanoparticles is degraded, leading to rapid drug release. Figure 2 F). The above characterization results demonstrate that the cell membrane-modified inflammatory-responsive release nanoparticles Ts@CPN@PM have been successfully prepared, exhibiting uniform particle size, good stability, and the ability to rapidly release drugs in an inflammatory microenvironment.

[0104] Experimental Example 1

[0105] In vitro antibacterial ability test

[0106] Test method:

[0107] (1) Take 10 μL of E. coli cryopreservation solution and add it to 10 mL of MH medium for overnight culture. The next day, dilute the logarithmic phase E. coli suspension with MH to OD. 600 The value is 0.08 (meaning the bacterial density is approximately 1.5 × 10⁸). 8 CFU mL -1 Then dilute with MH 300 times to obtain a bacterial density of 5×10⁻⁶. 5 CFU mL -1 Suspensions were prepared. 10 μL of polymyxin (PMB) at different concentrations, free Ts, and two drug-loaded nanoparticle solutions (Ts@CPN and Ts@CPN@PM) prepared in Example 1 (with final Ts concentrations of 0.5, 1, 2, 4, 8, 16, 32, 64, and 128 μg / mL) and 90 μL of a 5×10⁻⁶ m³ / well were added to a 96-well plate. 5 CFU mL -1 The bacterial suspension was prepared, and MMP-3 enzyme was added to a final concentration of 50 ng / mL to simulate the infection microenvironment. After incubation at 37°C for 16 h, the lowest drug concentration at which no bacterial growth was observed was recorded, which is the minimum inhibitory concentration (MIC) of the drug for that bacterium.

[0108] (2) Add 10 μL of E. coli cryopreservation solution to 10 mL of MH medium and incubate overnight. The next day, dilute the E. coli suspension with MH to OD. 600 The value was 0.07 (meaning the bacterial density was approximately 10). 8 CFU mL -1 Then dilute with MH 1000 times to obtain a bacterial density of 10. 5 CFU mL -1 A suspension of [amount missing]. 100 μL of 1 μg / mL PMB, free Ts, and solutions of the two drug-loaded nanoparticles (Ts@CPN and Ts@CPN@PM) prepared in Example 1 (diluted with MH) were added to a 96-well plate along with 100 μL of a [density missing] 100 μL [concentration missing]. 5 CFU mL -1 The bacterial suspension was incubated in a shaker at 37°C for 8 hours. The bacterial suspension was then spread onto MH agar plates and incubated for 18 hours. The colonies on the plates were photographed and counted.

[0109] (3) Take 10 μL of E. coli cryopreservation solution and add it to 10 mL of MH medium for overnight culture. The next day, dilute the E. coli suspension with MH to OD. 600 The concentration was 0.07, and then diluted with MH to a bacterial density of approximately 10. 5 CFU mL -1 One mL of bacterial suspension was co-incubated with one mL of 1 μg / mL PMB, free Ts, and the two drug-loaded nanoparticles (Ts@CPN and Ts@CPN@PM) prepared in Example 1 for 6 h. The bacteria were then collected by centrifugation, resuspended in physiological saline, and treated with 1 μL of propidium iodide (PI) and N-dimethylaniline N-oxide (DMAO) fluorescent dye, and incubated in the dark for 30 min. The bacterial staining was observed under a confocal laser scanning microscope; live bacteria exhibited green fluorescence, while dead bacteria exhibited both green and red fluorescence.

[0110] Test results are as follows Figure 3 As shown. Figure 3 The results of the in vitro antibacterial activity test were presented; among them, Figure 3 A represents the minimum inhibitory concentration (MIC) of polymyxin (PMB), free drug Ts, and two drug-loaded nanoparticles (Ts@CPN and Ts@CPN@PM) against E. coli. Figure 3 B and C are (B) bacterial plating images and (C) colony count results of E. coli after incubation of PMB, free drug Ts, and two drug-loaded nanoparticles (Ts@CPN and Ts@CPN@PM); Figure 3D and E are (D) live / dead staining confocal microscopy images and (E) mortality rate of bacteria after co-incubation of PMB, free drug Ts, and two drug-loaded nanoparticles (Ts@CPN and Ts@CPN@PM) with E. coli; .

[0111] Figure 3 A shows that the MIC of Ts@CPN@PM is half that of free Ts, comparable to that of the positive control drug PMB (clinically widely used to treat Gram-negative bacterial infections), indicating that the nanoparticles exhibit enhanced antibacterial activity compared to free drugs. Pre-stimulated macrophage membrane modification allows the nanoparticles to precisely target and tightly bind to the bacterial surface or surrounding area, forming localized drug concentration microregions, unlike free drugs which are uniformly dispersed in the culture medium. Simultaneously, the enzyme substrate switch within the nanoparticles is specifically cleaved by the MMP-3 enzyme, triggering a rapid and concentrated release of the drug on or around the bacterial surface, creating a transiently high-concentration drug region. Unlike the slow diffusion and continuous dilution of free drugs, this targeted aggregation and burst release effect allows the drug concentration at the bacterial site of action to rapidly reach the inhibitory concentration and maintain an effective duration of action, thereby reducing the total drug dose required to achieve the same antibacterial effect, directly manifested as a lower MIC value. Figure 3 Figures B and C show that, at the same drug concentration, Ts@CPN@PM treatment resulted in a significant reduction in E. coli colonies, indicating that Ts loaded with nanoparticles significantly improved the in vitro antibacterial effect. Figure 3 Figures D and E show that the Ts@CPN@PM treatment group had the highest bacterial mortality, indicating that Ts@CPN@PM nanoparticles have better antibacterial activity.

[0112] Experimental Example 2

[0113] In vitro targeting capability test

[0114] Test method: PM has the ability to target bacteria. To verify whether Ts@CPN@PM retains this property, coumarin 6 (Cou 6) labeled nanoparticles (Cou 6@CPN@PM, simply replace Ts with Cou 6) were co-incubated with Nile Red labeled E. coli and then detected by confocal laser scanning microscopy (CLSM).

[0115] Test results are as follows Figure 4 As shown. Figure 4 The results of in vitro targeting ability tests are presented; the figure shows confocal microscopy images and corresponding fluorescence intensity distribution maps of two fluorescent dye nanoparticles (Cou 6@CPN and Cou 6@CPN@PM) co-incubated with E. coli.

[0116] Figure 4The results showed that Cou 6@CPN@PM could bind better to E. coli, while retaining the ability of PM to target pathogens. Figure 4 The results showed that after co-incubating Cou 6@CPN@PM (green) with Nile Red-labeled E. coli (red), CLSM observation revealed that the red and green fluorescence overlapped well, resulting in yellow fluorescence, indicating significant co-localization. However, after co-incubating Cou 6@CPN (green) with Nile Red-labeled E. coli (red), almost no green fluorescence was observed on the bacterial surface. Fluorescence distribution curves were plotted using both red and green fluorescence signals, showing highly similar fluorescence trajectories in the Cou 6@CPN@PM group.

[0117] Experimental Example 3

[0118] In vivo targeting ability test

[0119] Ts@CPN@PM NPs showed good bacterial targeting ability in vitro, therefore, their targeting ability in vivo was further investigated.

[0120] Test Method: Two types of fluorescent dye-loaded nanoparticles (IR783@CPN and IR783@CPN@PM) were prepared according to the method in Example 1, replacing Ts with the fluorescent dye IR783. Healthy BALB / c mice were randomly divided into 6 groups: untreated free dye group (free IR783), untreated IR783@CPN group, untreated IR783@CPN@PM group, treated free dye group (free IR783), treated IR783@CPN group, and treated IR783@CPN@PM group, with 6 mice in each group. Mice in the treatment group were administered 50 μL of a 1×10⁻⁶ nanoparticle solution via endotracheal intubation. 7 CFU mL -1 E. coli bacterial culture was used to establish a model in mice. Mice in the non-model group were given an equal volume of physiological saline as a control. Two hours after treatment, mice in both groups were injected intravenously with different samples (dose of 50 mg IR783 / kg). Mice were sacrificed at 2, 12, 24, and 36 hours after administration, and major organs (heart, liver, spleen, lung, and kidney) were removed. After washing with PBS, in vitro tissue fluorescence imaging was performed, and fluorescence quantification was conducted on each tissue.

[0121] Test results are as follows Figure 5 As shown. Figure 5 The results of in vivo targeting ability testing were presented; among them, Figure 5A and B show the fluorescence imaging images and quantitative results of ex vivo tissues from unmodeled mice 2 h, 12 h, 24 h, and 36 h after tail vein injection of free fluorescent dye IR783 and two fluorescent dye-loaded nanoparticles (IR783@CPN and IR783@CPN@PM). Figure 5 C and D show the fluorescence imaging images (C) and quantitative results (D) of the ex vivo tissues of model mice 2 h, 12 h, 24 h, and 36 h after tail vein injection of free fluorescent dye IR783 and two fluorescent dye-loaded nanoparticles (IR783@CPN and IR783@CPN@PM).

[0122] Figure 5 The results showed that IR783@CPN@PM had significant lung-targeting properties in model mice and could maintain a high concentration in the lungs for a long time. Figure 5 AD). In unmodeled mice, the free IR783 group, IR783@CPN group, and IR783@CPN@PM group did not show rapid accumulation in the lungs at the beginning of drug administration, but instead showed relatively obvious liver targeting. As time went on, the drug was continuously metabolized to the liver, and the fluorescence intensity of the liver tissue continuously increased. Figure 5 (AB). In model mice, the IR783@CPN@PM group rapidly targeted the lungs after administration, with significant lung fluorescence accumulation appearing 2 hours post-injection, accounting for nearly 70% of the total fluorescence, and maintaining this for a period of time. 36 hours post-injection, IR783@CPN@PM was rapidly cleared from most major organs except the liver. In contrast, free IR783 and IR783@CPN were mainly distributed in the liver (AB). Figure 5 CD).

[0123] Combination Figure 4 and Figure 5 The results indicate that PM modification enables Ts@CPN@PM to effectively target pathogens and infected tissues. This will facilitate the delivery of antimicrobial drugs from the bloodstream to the site of bacterial infection, achieving efficient delivery of antimicrobial drugs and avoiding degradation of antimicrobial peptides in the body, which could lead to decreased efficacy or biotoxicity from high-dose administration.

[0124] Test Example 4

[0125] In vivo therapeutic performance test

[0126] Test method: Healthy BALB / c mice were endotracheally intubated and given 50 μL of a 1×10⁻⁶ solution. 7 CFU mL -1Two hours after infection with E. coli culture, mice were randomly divided into six groups according to different treatment methods: Model group (PBS, 100 μL), PMB group (4 mg / kg, 100 μL), Ts group (10 mg / kg, 100 μL), Ts@CPN group (Ts concentration 10 mg / kg, 100 μL), Ts@CPN@PM group (Ts concentration 10 mg / kg, 100 μL), and Negative control group (no infection modeling, no drug administration), with 15 mice in each group. The corresponding drug was administered via tail vein. Twenty-four hours after drug administration, five mice from each group were sacrificed, and bronchoalveolar lavage fluid (BALF) and lung tissue were extracted. BALF was obtained from the left lung; the upper right lobe was used for tissue smearing and colony counting, and the lower right lobe was used for histopathological examination. A small incision was made in the trachea, and 0.3 mL of PBS buffer was instilled into the trachea using a catheter. This step was repeated three times to obtain BALF. The remaining 10 mice in each group underwent survival analysis. BALF (Basal Body Fluid) was centrifuged at 2000 rpm for 10 min, and the levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) in the BALF supernatant were detected using enzyme-linked immunosorbent assay (ELISA). The right upper lobe of the lung was washed with physiological saline, weighed, and immersed in a certain amount of physiological saline. It was then homogenized using a high-pressure cell homogenizer in an ice bath to obtain a suspension. This suspension was diluted with physiological saline and spread onto MH agar plates. After 18 h of incubation, the plates were photographed and colonies were counted. The right lower lobe of the lung was washed with physiological saline, stained with hematoxylin and eosin (H&E), and observed under a microscope.

[0127] Test results are as follows Figure 6 As shown. Figure 6 The results of in vivo therapeutic performance tests were presented; among them, Figure 6 A is a diagram of the in vivo treatment plan; Figure 6 B represents the survival curve results; Figure 6 C represents the result of weight change; Figure 6 D and E are the lung tissue smear images (D) and colony count results (E); Figure 6 F represents the protein level detection result in bronchoalveolar lavage fluid (BALF); Figure 6 GI refers to the detection results of (G) tumor necrosis factor α (TNF-α), (H) interleukin 1β (IL-1β) and (I) interleukin levels in BALF; Figure 6 J represents the results of hematoxylin-eosin (H&E) staining of lung tissue; difference analysis compared with the Model group: .

[0128] Figure 6The results showed that Ts@CPN@PM had good in vivo therapeutic effects. Figure 6 BJ). Figure 6 B showed that the survival rate of mice in the Ts@CPN@PM treatment group (60%) was higher than that in the Model group (10%), Ts (30%), and Ts@CPN (50%) treatment groups, and comparable to that in the PMB group. Figure 6 C showed that after treatment with Ts@CPN@PM, the body weight of infected mice gradually returned to normal. Lung tissue smear results showed that Ts@CPN@PM treatment significantly reduced the bacterial load ( Figure 6 DE). Inflammatory responses typically lead to increased vascular permeability, causing plasma proteins to leak into the alveolar spaces and pulmonary interstitium, resulting in elevated protein levels in lung tissue. The Model group mice showed extremely high protein levels in their BALF, while the Ts@CPN@PM group mice showed significantly lower protein levels in their BALF, approaching those of the Negative control group, indicating that pulmonary edema and interstitial fluid leakage were effectively alleviated in the mice. Figure 6 F). Compared with other groups, the levels of pro-inflammatory cytokines TNF-α, IL-6, and IL-1β in the BALF of mice in the Ts@CPN@PM group were significantly reduced (F). Figure 6 GI). H&E staining of lung tissue showed that Ts@CPN@PM significantly alleviated the symptoms of bacterial lung inflammation (such as inflammatory cell infiltration, alveolar wall widening, bronchiolar mucosal necrosis, and inflammatory exudate in the alveolar cavities). After treatment, the lung tissue structure was intact, the alveolar cavities were clear and free of exudate, and pulmonary edema and inflammatory cell infiltration were reduced. Figure 6 J).

[0129] Experimental Example 5

[0130] In vitro safety testing

[0131] Test method:

[0132] (1) Whole blood from BALB / c mice was collected in an anticoagulant tube, centrifuged at 1000 rpm for 10 min to collect the erythrocyte pellet, and resuspended in physiological saline (NE). This operation was repeated 5 times to prepare a 2% erythrocyte suspension with NE. An equal volume of the test drug solution (PMB, free drug Ts, Ts@CPN, and Ts@CPN@PM) was added to the erythrocyte suspension. The final concentrations of PMB or Ts were 3.125, 6.25, 12.5, 25, 50, 100, 200, 400, and 800 μg / mL. NE was set as a negative control, and ultrapure water was set as a positive control. After incubation in a 37℃ water bath, the mixture was centrifuged at 1000 rpm for 10 min, photographed, and the supernatant was collected to determine the OD. 540 The absorbance value. The formula for calculating the hemolysis rate is as follows:

[0133]

[0134] (2) Human alveolar basal epithelial cells A549 (lung cancer), human normal lung epithelial cells BEAS 2B, and mouse mononuclear macrophage leukemia cells RAW 264.7 were cultured to the logarithmic growth phase and then subjected to 2×10⁻⁶ cells per cell line. 4 Seeds were planted at a density of 100 cells / well in 96-well plates. After incubation to a density of 60-70%, the test solutions (PMB, free drug Ts, Ts@CPN, and Ts@CPN@PM) were added at final concentrations of PMB or Ts of 6.25, 12.5, 25, 50, 100, and 200 μg / mL. A blank medium was set up as a blank control. After incubation for 24 h, 10 μL of CCK-8 working solution was added, and the plates were incubated in the dark for 1 h. The OD was then measured. 450 The absorbance value. The formula for calculating cytotoxicity is as follows:

[0135]

[0136] (3) Human alveolar basal epithelial cells A549 (lung cancer), human normal lung epithelial cells BEAS 2B, and mouse mononuclear macrophage leukemia cells RAW 264.7 were cultured to the logarithmic growth phase and then cultured at 3×10⁻⁶ cells / year. 4 Seeds were placed at a density of 1 cell / dish in confocal microscopy dishes and cultured until the density reached 60-70%. Then, the test solutions (PMB, free drug Ts, Ts@CPN NPs, and Ts@CPN@PMNPs) were added, with a final PMB or Ts concentration of 25 μg / mL. A blank culture medium was set up as a blank control. After incubation for 24 h, the drug solutions and blank culture medium were discarded. Calcein / PI staining working solution was added, and the cells were incubated in the dark for 30 min. Observation was then performed under a confocal laser scanning microscope. Live cells showed green fluorescence, while dead cells showed red fluorescence.

[0137] Test results are as follows Figure 7 As shown. Among them, Figure 7 A represents the hemolytic toxicity of PMB, free drug Ts, and two drug-loaded nanoparticles (Ts@CPN and Ts@CPN@PM) on blood cells at different drug concentrations; Figure 7 B represents the toxicity test results of PMB, free drug Ts, and two drug-loaded nanoparticles (Ts@CPN and Ts@CPN@PM) on A549, BEAS 2B, and RAW 264.7 cells at different drug concentrations; Figure 7 CE represents confocal microscopy images of live / dead cells after co-incubation with PMB, free drug Ts, and two drug-loaded nanoparticles (Ts@CPN and Ts@CPN@PM) and (C) A549, (D) BEAS 2B, and (E) RAW 264.7 cells. Difference analysis compared with NE: ns, no significant difference.

[0138] Figure 7 The results showed that Ts@CPN@PM exhibited good in vitro biocompatibility, low hemolysis rate against mammalian erythrocytes, low toxicity to normal cells, and good safety. Among these, Figure 7 Results showed that even at drug concentrations far exceeding therapeutic doses (800 μg / mL), Ts@CPN@PM did not exhibit hemolytic toxicity to erythrocytes. When the Ts concentration reached 200 μg / mL, Ts@CPN@PM still showed low cytotoxicity against A549, BEAS 2B, and RAW 264.7 cells, with cell viability reaching 95%, indicating that Ts@CPN@PM possesses extremely low cytotoxicity. Figure 7 BE).

[0139] Experimental Example 6

[0140] In vivo safety testing

[0141] Test method:

[0142] Eight-week-old healthy male ICR mice were used in the experiment and randomly divided into five groups according to different treatment methods: Control group (PBS, 100 μL), PMB group (10 mg / kg, 100 μL), Ts group (10 mg / kg, 100 μL), Ts@CPN group (Ts concentration 10 mg / kg, 100 μL), and Ts@CPN@PM group (Ts concentration 10 mg / kg, 100 μL), with five mice in each group. The corresponding drugs were administered via tail vein. Drugs were administered once daily for seven consecutive days, and mouse weight and survival status were recorded daily. At the end of the experiment, orbital blood samples were collected from the mice to detect routine blood parameters (such as red blood cells, white blood cells, and platelets) and blood biochemical markers representing liver and kidney function (including alanine aminotransferase, aspartate aminotransferase, albumin, blood urea nitrogen, creatinine, and uric acid). Simultaneously, histological sections and hematoxylin and eosin (H&E) staining were performed on the major organs (heart, liver, spleen, lungs, and kidneys).

[0143] Test results are as follows Figure 8 As shown. Among them, Figure 8 A shows the changes in body weight of mice in each group after 7 days of continuous injection of PBS, PMB, Ts, Ts@CPN and Ts@CPN@PM; Figure 8Figure BD shows the routine blood parameters of mice in each group after continuous injection of PBS, PMB, Ts, Ts@CPN, and Ts@CPN@PM for 7 days: (B) red blood cells, (C) white blood cells, and (D) platelets; Figure EJ shows the blood biochemical parameters of mice in each group after continuous injection of PBS, PMB, Ts, Ts@CPN, and Ts@CPN@PM for 7 days: (E) alanine aminotransferase (ALT), (F) aspartate aminotransferase (AST), (G) albumin (ALB), (H) blood urea nitrogen (BUN), (I) creatinine (CRE), and (J) uric acid (UA); Figure K shows the H&E staining results of major organs (heart, liver, spleen, lung, and kidney) of mice in each group after continuous injection of PBS, PMB, Ts, Ts@CPN, and Ts@CPN@PM for 7 days; Difference analysis compared with the Control group: ns, no significant difference; .

[0144] Figure 8 The results showed that Ts@CPN@PM had good biocompatibility in vivo, and continuous intravenous administration for 7 days had no significant toxicity to the body weight, quality of life, hematological parameters, blood biochemical parameters, and major organs of healthy mice. Figure 8 Results showed that after 7 days of continuous intravenous administration, except for the PMB group which showed a decrease in body weight, the other groups of mice all showed normal weight gain, indicating a good quality of life. Figure 8 The BD results showed that the number of white blood cells in the PMB group mice was significantly increased, while the hematological parameters (red blood cells, white blood cells, and platelets) of the other groups of mice were normal. Figure 8 EJ results showed that continuous administration of PMB led to impaired liver and kidney function in mice, specifically manifested as abnormal blood biochemical indicators. The indicators in the other treatment groups showed no significant differences compared to the Control group. H&E staining further revealed significant pathological damage in the major organs (especially the kidneys and liver) of the PMB group mice: significant swelling and necrosis of renal tubular epithelial cells, accompanied by brush border shedding, tubular dilation, protein casts, inflammatory cell infiltration, and interstitial edema; disordered hepatic cords, hepatocyte edema and degeneration, and focal inflammatory infiltration in the liver; disordered spleen tissue structure, blurred boundaries between red and white pulp, accompanied by inflammatory cell infiltration and local congestion; and extensive inflammatory cell infiltration and alveolar structural destruction observed in the lung tissue, manifested as uneven alveolar cavity size and distorted shape, partial alveolar wall rupture and fusion, and loss of normal septal structure. However, mice in other treatment groups did not show tissue damage, necrosis, or inflammatory infiltration. Figure 8 K).

Claims

1. A cell membrane-modified inflammatory-responsive release nanoparticle, characterized in that, The nanoparticles have a polylactic acid-glycolic acid copolymer (PLGA) core and their surface is modified with an immune cell membrane; wherein the polylactic acid-glycolic acid copolymer is loaded with an antibacterial drug and is also modified with a positively charged material and a responsive switch; the immune cell membrane is a cell membrane obtained by isolating immune cells pre-stimulated by pathogens.

2. The cell membrane-modified inflammatory-responsive release nanoparticles according to claim 1, characterized in that, The antibacterial drug is preferably an antibacterial polypeptide drug.

3. The cell membrane-modified inflammatory-responsive release nanoparticles according to claim 1, characterized in that, The positively charged material includes any one or more of polyvinyl alcohol, polysorbate, sodium lauryl sulfate, chitosan, gelatin, and gum arabic.

4. The cell membrane-modified inflammatory-responsive release nanoparticles according to claim 1, characterized in that, The responsive switch includes any one or more of the following: MMP-2 protease substrate, MMP-3 protease substrate, MMP-9 protease substrate, elastase substrate, esterase substrate, β-lactamase substrate, neuraminidase substrate, polyhistidine, polyacrylic acid, polysulfide, polyselenide, metal-organic framework, and anti-LPS antibody.

5. The cell membrane-modified inflammatory-responsive release nanoparticles according to claim 1, characterized in that, The immune cells include any one or more of macrophages, T lymphocytes, B lymphocytes, dendritic cells, natural killer cells, neutrophils, mast cells, eosinophils, and basophils.

6. The cell membrane-modified inflammatory-responsive release nanoparticles according to claim 1, characterized in that, The pathogens include any one or more of Escherichia coli, Salmonella, Shigella, Pseudomonas aeruginosa, Haemophilus influenzae, Klebsiella pneumoniae, Acinetobacter baumannii, and Neisseria gonorrhoeae.

7. A method for preparing cell membrane-modified inflammatory-responsive release nanoparticles according to claim 1, characterized in that, The steps include the following: (1) Synthesis of drug-loaded nanoparticles a. Dissolve the carrier material PLGA in a solvent and stir to obtain an organic phase; b. Dissolve the antibacterial drug and the responsive switch in water and stir to obtain an aqueous phase; c. Add the aqueous phase to the organic phase and stir to react and obtain the primary emulsion; d. The promulgated material is added to an external aqueous solution containing a positively charged material, stirred to react, washed, and dried to obtain nanoparticles; (2) Preparation of cell membrane vesicles pre-stimulated by pathogens e. Culture immune cells to the logarithmic phase; f. Add pathogens to stimulate the body; g. After centrifuging to collect cells, perform sonication and centrifugation, collect the supernatant and centrifuge again to obtain cell membrane vesicles; (3) Modification of drug-loaded nanoparticles The nanoparticles obtained in step d were dissolved in water, mixed with the cell membrane vesicles obtained in step g, and then sonicated to obtain cell membrane-modified inflammatory-responsive nanoparticles.

8. The use of the cell membrane-modified inflammatory-responsive release nanoparticles of claim 1 in the preparation of antibacterial drugs.

9. The application according to claim 8, characterized in that, Application of the cell membrane-modified inflammatory-responsive release nanoparticles in the preparation of drugs for treating bacterial infections and related diseases caused by bacterial infections.

10. The use of the cell membrane-modified inflammatory-responsive release nanoparticles of claim 1 in the in vivo delivery of cationic peptide drugs.

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