A nanomedicine delivery system targeting neutrophils, its preparation method and application

Abstract

This invention relates to a nanomedicine delivery system targeting neutrophils, its preparation method, and its application. The nanomedicine delivery system is prepared by the following method: S1, preparing liposome 1 encapsulating drug 1; S2, preparing liposome 2 encapsulating drug 2; S3, mixing and incubating the liposome 1 and liposome 2 encapsulating drug 1 and drug 2, and connecting the corresponding ligands and receptors in liposome 1 and liposome 2 to obtain the nanomedicine delivery system; the ligands and receptors are biotin and streptavidin, respectively. This invention successfully connects two drug liposomes with different functions through a ligand and receptor system to obtain the nanomedicine delivery system, which can enhance the chemotaxis of neutrophils to the site of infection and improve the antibacterial properties of NETs, ​​exhibiting very good anti-infective effects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a nanomedicine delivery system targeting neutrophils, its preparation method, and its application. Background Technology

[0002] Neutrophils can migrate to the site of infection in response to chemokine induction and release neutrophil extracellular traps (NETs) composed of histones, granules, elastase, myeloperoxidase, and DNA to capture and eliminate pathogens invading the body. However, in severe infectious diseases such as sepsis, continuous stimulation by intracellular and extracellular pathogens leads to internalization of the neutrophil surface chemokine receptor CXCR2, weakening its lesion chemotaxis. Furthermore, the bactericidal effect of NETs is insufficient; some pathogens can degrade NETs by forming nucleases, thus escaping NET capture and killing. Therefore, NETs alone are insufficient to meet anti-infection requirements.

[0003] Currently, nanomedicines designed based on the natural chemotaxis of neutrophils to lesion sites are widely studied. The delivery of nanomedicines using neutrophils generally follows two different strategies. The first involves loading extracted neutrophils with nanomedicines in vitro. However, due to the short lifespan of neutrophils, complex operations can severely affect neutrophil viability and pose a risk of in vitro contamination, making this strategy impractical. The second strategy involves hijacking neutrophils with nanomedicines in systemic circulation. This strategy avoids the shortcomings of the first approach, but requires nanoparticles to have the ability to bind efficiently to neutrophils. Furthermore, because neutrophils cannot respond to chemokines and migrate to the infection site during severe infections, pathogens cannot be cleared and spread rapidly, leading to disease exacerbation. Therefore, "re-education" measures for neutrophil lesion chemotaxis are crucial for the treatment of related diseases. Current main measures involve intervening in the internalization-related signaling pathway of the neutrophil surface chemokine receptor CXCR2. Related studies have shown that neutrophil NETs can release drugs from the neutrophil cytoplasm to the extracellular space, such as releasing antimicrobial peptides contained in the neutrophil itself to clear extracellular pathogens; or NETs can be recognized and engulfed by macrophages to clear intracellular parasites of macrophages.

[0004] Currently, the goal is to develop a drug that can effectively restore the chemotaxis of neutrophils at the site of infection and enhance the antibacterial activity of NETs, ​​thereby effectively eliminating intracellular and extracellular pathogens. Summary of the Invention

[0005] Based on this, the purpose of this invention is to provide a nanomedicine delivery system that targets neutrophils. This nanomedicine delivery system can both hijack and educate neutrophils to restore their chemotaxis to the site of infection, and enhance the anti-intracellular and extracellular bacteria effect of neutrophil NETs, ​​thereby achieving better therapeutic effects.

[0006] The first aspect of this invention is to provide a method for preparing a nanomedicine delivery system targeting neutrophils, comprising the following steps:

[0007] S1. Lecithin, cholesterol, poly(2-methacryloyloxyethylphosphorylcholine) modified fatty amine, histidine modified fatty amine, ligand or receptor are added to organic solvent 1 to prepare liposome 1; while stirring, a solution containing drug 1 is dropped into the liposome 1 to obtain liposome 1 encapsulated with drug 1; the drug 1 is an anti-inflammatory drug.

[0008] S2. Lecithin, cholesterol, dioleoylphosphatidylethanolamine, drug 2, and a receptor or ligand corresponding to the ligand or receptor added in step S1 are added to organic solvent 2 to prepare liposome 2; the liposome 2 is mixed with platelet membrane to prepare liposome 2 encapsulated with drug 2; the drug 2 is a drug that induces neutrophils to chemotaxis toward the site of infection.

[0009] S3. The liposome 1 encapsulating drug 1 and the liposome 2 encapsulating drug 2 are mixed and incubated, and the corresponding ligands and receptors in the liposome 1 and liposome 2 are connected to obtain the drug 1.

[0010] The ligand and receptor are biotin and streptavidin, respectively.

[0011] In some embodiments, streptavidin is added in step S1 and biotin is added in step S2.

[0012] In some embodiments, the mass ratio of lecithin to streptavidin in step S1 is (150-200):(0.5-3), preferably 150:(0.5-3), and more preferably 150:(0.5-1.5).

[0013] In some embodiments, the mass ratio of lecithin to biotin in step S2 is (75-100):(1-5), preferably 75:(1-5), and more preferably 75:(1-3).

[0014] In some embodiments, drug 1 is selected from at least one of itaconic acid and antibiotics; drug 2 is selected from at least one of GRK2 inhibitors and ERK inhibitors.

[0015] In some embodiments, drug 1 is itaconic acid and drug 2 is fenofibrate.

[0016] In some embodiments, the mass ratio of lecithin to itaconic acid in step S1 is (150-200):(9-45), preferably 150:(15-45), and more preferably 150:(30-45).

[0017] In some embodiments, the mass ratio of lecithin to fenofibrate in step S2 is (75-100):(20-30), preferably 75:(20-30).

[0018] In some embodiments, in step S3, the liposomes 1 encapsulating drug 1 and the liposomes 2 encapsulating drug 2 are mixed and incubated at a mass ratio of (6-30):(20-30) of drug 1 to drug 2.

[0019] In some embodiments, the poly(2-methacryloyloxyethyl phosphorylcholine) modified fatty amine is selected from poly(2-methacryloyloxyethyl phosphorylcholine) modified octadecylamine, poly(2-methacryloyloxyethyl phosphorylcholine) modified stearoylphosphatidylethanolamine, poly(2-methacryloyloxyethyl phosphorylcholine) modified hydrogenated soybean phosphatidylcholine, and poly(2-methacryloyloxyethyl phosphorylcholine) modified dipalmitoylphosphatidylcholine.

[0020] In some embodiments, the histidine-modified fatty amine is selected from histidine-modified octadecylamine, histidine-modified stearoylphosphatidylethanolamine, histidine-modified hydrogenated soybean phosphatidylcholine, and histidine-modified dipalmitoylphosphatidylcholine.

[0021] In some embodiments, the poly(2-methacryloyloxyethyl phosphorylcholine) modified fatty amine is selected from poly(2-methacryloyloxyethyl phosphorylcholine) modified octadecylamine, and the histidine modified fatty amine is selected from histidine modified octadecylamine.

[0022] In some embodiments, the mass ratio of lecithin, cholesterol, poly(2-methacryloyloxyethylphosphorylcholine) modified octadecylamine, and histidine modified octadecylamine in step S1 is (150-200): 50: (10-20): (10-20).

[0023] In some embodiments, the mass ratio of lecithin, cholesterol, and dioleoylphosphatidylethanolamine in step S2 is (75-100):25:(10-20).

[0024] In some embodiments, the mass ratio of lecithin to total platelet membrane protein in step S2 is 1:(50-500), preferably 1:(50-250), and more preferably 1:(50-100).

[0025] A second aspect of the present invention is to provide a nanomedicine delivery system targeting neutrophils, which is prepared by the preparation method described above.

[0026] A third aspect of the present invention is the application of the nanomedicine delivery system for targeting neutrophils as described above in the preparation of anti-infective drugs.

[0027] A fourth aspect of the present invention is to provide an anti-infective drug comprising an active ingredient and a pharmaceutically acceptable carrier; said active ingredient comprising a nanomedicine delivery system targeting neutrophils as described above.

[0028] This invention provides a nanomedicine delivery system targeting neutrophils, which successfully connects two drug liposomes with different functions (liposome 1 encapsulating drug 1 and liposome 2 encapsulating drug 2) through a ligand and receptor system to obtain the nanomedicine delivery system. Liposome 2 encapsulating drug 2 is hybridized with the platelet membrane, allowing the nanomedicine delivery system to be recognized and phagocytosed by neutrophils. After endocytosis into neutrophils, liposome 2 encapsulating drug 2 fuses with the neutrophil lysosomal membrane in the acidic environment of lysosomes, releasing drug 2 and enhancing the chemotaxis of neutrophils to lesions. Meanwhile, liposome 1 encapsulating drug 1 enters the neutrophil cytoplasm intact, is delivered by neutrophils to the site of infection, and is released through NETs, ​​enhancing the intracellular and extracellular antibacterial effects of NETs. Furthermore, the release of drug 1 further activates the body's innate immune system, thereby further enhancing the chemotaxis of neutrophils to the site of infection.

[0029] This invention successfully prepared a nanomedicine delivery system comprising two liposomes with different functional drugs via a ligand-receptor linkage system. Furthermore, through extensive experiments and research, the inventors discovered that the mass ratio of lecithin to ligand and receptor in the liposomes significantly influences the efficient and successful linkage of the two drug liposomes via ligand and receptor, as well as the stability of the resulting nanomedicine delivery system. A suitable mass ratio of lecithin to ligand and receptor was screened to ensure the successful preparation of the nanomedicine delivery system and its therapeutic efficacy in vivo. Further, the ratio of drug 1 to drug 2 has a significant impact on the anti-infective effect of the nanomedicine delivery system. An appropriate mass ratio of drug 1 to drug 2 in the nanomedicine delivery system can simultaneously enhance the chemotaxis and bactericidal properties of neutrophils to the site of infection, thereby giving the nanomedicine delivery system of this invention a very good anti-infective effect. Attached Figure Description

[0030] Figure 1 This is the proton NMR spectrum of pMPC-ODA.

[0031] Figure 2 This is the hydrogen NMR spectrum of His-ODA.

[0032] Figure 3 The particle size and zeta potential of ITA-HpLipos are given.

[0033] Figure 4 Results of platelet membrane hybridization were used to screen for platelet membrane hybridization ratios using flow cytometry.

[0034] Figure 5 The particle size and zeta potential of Fen-PDLipos are given.

[0035] Figure 6 This is a representative transmission electron microscope image of the IF-CLipos.

[0036] Figure 7 This is a representative fluorescence image obtained by laser confocal microscopy of IF-CLipos.

[0037] Figure 8 Flow cytometry was used to investigate the results of IF-CLipos hijacking neutrophils in vivo.

[0038] Figure 9 The particle size and zeta potential of IF-CLipos under different pH conditions are shown.

[0039] Figure 10 To investigate the upregulation effect of IF-CLipos on the CXCR2 receptor on the surface of neutrophils using immunofluorescence.

[0040] Figure 11 Neutrophils release ITA-HpLipos via NETs to clear extracellular bacteria.

[0041] Figure 12 Neutrophils release ITA-HpLipos via NETs to clear intracellular bacteria.

[0042] Figure 13 The results show the detection of neutrophil infiltration in intestinal infection lesions in mice.

[0043] Figure 14 The results show the bacterial content in the mouse intestines. Detailed Implementation

[0044] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0045] Unless otherwise specified, experimental methods in the following examples are generally performed under standard conditions or as recommended by the manufacturer. All commonly used chemical reagents used in the examples are commercially available products.

[0046] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this invention includes any and all combinations of one or more of the associated listed items.

[0047] Furthermore, as used herein, the term "or" is an inclusive "or" sign and is equivalent to the term "and / or" unless the context clearly specifies otherwise. The term "based on" is not exclusive and allows for basing on other factors not described unless the context clearly specifies otherwise. Additionally, throughout the specification, the meanings of "an," "a," and "the" include plural indicators. The meaning of "in" includes both "in" and "on."

[0048] Some embodiments of the present invention relate to a method for preparing a nanomedicine delivery system targeting neutrophils, comprising the following steps:

[0049] S1. Lecithin, cholesterol, poly(2-methacryloyloxyethylphosphorylcholine) modified fatty amine, histidine modified fatty amine, ligand or receptor are added to organic solvent 1 to prepare liposome 1; while stirring, a solution containing drug 1 is dropped into the liposome 1 to obtain liposome 1 encapsulated with drug 1; the drug 1 is an anti-inflammatory drug.

[0050] S2. Lecithin, cholesterol, dioleoylphosphatidylethanolamine, drug 2, and a receptor or ligand corresponding to the ligand or receptor added in step S1 are added to organic solvent 2 to prepare liposome 2; the liposome 2 is mixed with platelet membrane to prepare liposome 2 encapsulated with drug 2; the drug 2 is a drug that induces neutrophils to chemotaxis toward the site of infection.

[0051] S3. The liposome 1 encapsulating drug 1 and the liposome 2 encapsulating drug 2 are mixed and incubated, and the corresponding ligands and receptors in the liposome 1 and liposome 2 are connected to obtain the drug 1.

[0052] The ligand and receptor are biotin and streptavidin, respectively.

[0053] This invention successfully connects two drug liposomes with different functions (liposome 1 encapsulating drug 1 and liposome 2 encapsulating drug 2) through a ligand and receptor system to obtain the nanodrug delivery system.

[0054] Through extensive experiments and research, the inventors discovered that the mass ratio of lecithin to ligand and receptor in liposomes has a significant impact on the successful conjugation of two drug liposomes and the stability of the resulting nanomedicine delivery system. They screened and determined suitable mass ratios for lecithin to ligand and receptor: when streptavidin is added in step S1 and biotin is added in step S2, the mass ratio of lecithin to streptavidin in step S1 is (150–200):(0.5–3); and the mass ratio of lecithin to biotin in step S2 is (75–100):(1–5). These lecithin-to-ligand-receptor mass ratios enable successful and efficient conjugation of drug-encapsulated liposome 1 and drug-encapsulated liposome 2 via ligand and receptor, obtaining the nanomedicine delivery system and ensuring its stability, allowing it to exert its intended effect upon entering the body.

[0055] Furthermore, based on the successful preparation and stability of the nanomedicine delivery system of the present invention, by further controlling the ratio of drug 1 and drug 2 in the above-mentioned nanomedicine delivery system, the chemotaxis and bactericidal properties of neutrophils towards the site of infection can be simultaneously enhanced. The inventors have discovered that when drug 1 is itaconic acid and drug 2 is fenofibrate, the mass ratio of lecithin to itaconic acid in step S1 is (150-200):(9-45), and the mass ratio of lecithin to fenofibrate in step S2 is (75-100):(20-30), which can effectively improve the antibacterial effect of the nanomedicine delivery system of the present invention, giving it a very good anti-infective effect.

[0056] The liposomes 1 encapsulating drug 1 in the nanomedicine delivery system of this invention are prepared using raw materials including poly(2-methacryloyloxyethylphosphorylcholine) modified fatty amines and histidine modified fatty amines. This allows the surface of the prepared liposomes 1 encapsulating drug 1 to be modified with poly(2-methacryloyloxyethylphosphorylcholine) (pMPC) and histidine (His). When the liposomes 1 encapsulating drug 1 enter neutrophils, they escape as lysosomes through the histidine modification on their surface, entering the neutrophil cytoplasm intact. They are then delivered by the neutrophils to the site of infection and released via NETs. The positively charged pMPC modification interacts with the negatively charged LPS on the surface of the pathogen via electrostatic interaction, promoting the pathogen's uptake and release of drug 1, thus enhancing the intracellular and extracellular antibacterial effects of NETs.

[0057] The poly(2-methacryloyloxyethylphosphatidylcholine)-modified fatty amine and histidine-modified fatty amine can be obtained by modifying the fatty amines conventionally used in the preparation of liposomes, or commercially available products can be purchased. Both methods can be used to prepare the nanomedicine delivery system of the present invention. For example, in some embodiments, the poly(2-methacryloyloxyethylphosphatidylcholine)-modified fatty amine is selected from poly(2-methacryloyloxyethylphosphatidylcholine)-modified octadecylamine (pMPC-ODA), poly(2-methacryloyloxyethylphosphatidylcholine)-modified stearoylphosphatidylethanolamine (pMPC-DSPE), poly(2-methacryloyloxyethylphosphatidylcholine)-modified hydrogenated soybean phosphatidylcholine, and poly(2-methacryloyloxyethylphosphatidylcholine)-modified dipalmitoylphosphatidylcholine. The histidine-modified fatty amine is selected from histidine-modified octadecylamine, histidine-modified stearoylphosphatidylethanolamine, histidine-modified hydrogenated soybean phosphatidylcholine, and histidine-modified dipalmitoylphosphatidylcholine. Among them, poly(2-methacryloyloxyethylphosphorylcholine) modified octadecylamine and histidine modified octadecylamine are inexpensive and can effectively reduce preparation costs, so they can be preferred.

[0058] The structural formula of the poly(2-methacryloyloxyethylphosphorylcholine)-modified octadecylamine is shown in Formula I below:

[0059]

[0060] Where X is selected from halogens; n≥4, and the average degree of polymerization is 5.

[0061] In some of these embodiments, X is selected from Br, Cl, and I.

[0062] In some embodiments, the structure of the pMPC-ODA is as follows:

[0063]

[0064] The pMPC-ODA is obtained by reacting 2-methacryloyloxyethyl phosphorylcholine (MPC) and octadecylamine (ODA), and the reaction formula (X is selected from Br) is as follows:

[0065]

[0066] pMPC-ODA can be prepared using conventional techniques in the art. For example, in some embodiments, pMPC-ODA is prepared by the following method: dispersing ODA in a solvent, then adding 2-bromoisobutyryl bromide dropwise into the mixture, stirring the reaction, washing, removing the solvent, and obtaining an intermediate product; taking the intermediate product and MPC and adding them to a solvent, purging nitrogen gas to remove oxygen, adding CuBr in the dark, stirring and reacting in the dark to obtain pMPC-ODA.

[0067] In some embodiments, the mass ratio of ODA to MPC is (1-2):(1.5-3)1.51:1.94.

[0068] In some embodiments, the reaction temperature is 30°C to 50°C, preferably 35°C to 45°C, more preferably 40°C; and the reaction time is 10h to 12h.

[0069] The structural formula of the histidine-modified octadecylamine is shown in Formula II below:

[0070]

[0071] The His-ODA is obtained by the reaction of Boc-His(Boc)-OH with ODA, as shown in the following reaction formula:

[0072]

[0073] His-ODA can be prepared using conventional techniques in the art. For example, in some embodiments, the His-ODA is prepared by the following method: dissolving Boc-His(Boc)-OH, ODA, a polypeptide condensing agent, and N,N-diisopropylethylamine (DIPEA) in a solvent, stirring the reaction, vacuum filtering, collecting the solid precipitate, drying, dissolving, separating by column chromatography, removing the organic solvent, and obtaining Boc-His(Boc)-ODA; adding the Boc-His(Boc)-ODA to dioxane hydrochloride, stirring the reaction, adjusting the pH of the reaction mixture, and vacuum filtering to obtain His-ODA.

[0074] In some embodiments, the mass ratio of Boc-His(Boc)-OH, ODA, polypeptide condensing agent and DIPEA is 1:(0.1-1):(1-2):(0.1-1).

[0075] In some embodiments, the pH value is 7.5 to 8.5.

[0076] In some embodiments, the polypeptide condensing agent is 2-(7-azabenzotriazole)-N,N,N′,N′-tetramethylurea hexafluorophosphate.

[0077] In some embodiments, the solvent is N,N'-dimethylformamide.

[0078] In some embodiments, the preparation method of the nanomedicine delivery system targeting neutrophils includes the following steps:

[0079] S1. Lecithin, cholesterol, poly(2-methacryloyloxyethylphosphorylcholine) modified fatty amine, histidine modified fatty amine, ligand or receptor are added to organic solvent 1 and completely dissolved. Organic solvent 1 is removed to form a film. Ultrapure water is added for hydration to obtain primary liposomes. The liposomes are ultrasonically broken up and filtered to obtain liposome 1. While stirring, a solution containing drug 1 is dripped into the liposome 1 to obtain liposome 1 encapsulated with drug 1.

[0080] S2. Lecithin, cholesterol, dioleoylphosphatidylethanolamine, drug 2, and the receptor corresponding to the ligand added in step S1 or the ligand corresponding to the added receptor are completely dissolved in organic solvent 2. The organic solvent 2 is removed to form a thin film. Ultrapure water is added for hydration to obtain primary liposomes. The liposomes are ultrasonically broken up and filtered to obtain liposome 2. The liposome 2 is mixed with a platelet membrane, ultrasonically dispersed, and then the mixed solution is extruded through a membrane to obtain liposomes 2 encapsulating drug 2.

[0081] S3. The liposome 1 encapsulating drug 1 and the liposome 2 encapsulating drug 2 are mixed and incubated, and the corresponding ligands and receptors in the liposome 1 and liposome 2 are connected to obtain the drug.

[0082] In some embodiments, the organic solvent 1 includes chloroform and methanol; preferably, the volume ratio of chloroform to methanol is (1-3):1.

[0083] In some embodiments, the organic solvent 2 includes oleic acid and chloroform; preferably, the volume ratio of oleic acid to chloroform is (0.005-0.02):15.

[0084] In some embodiments, the ultrasonic fragmentation conditions in step S1 are: power 195W to 325W, time 5min to 7min, ultrasonic 1s to 3s, pause 2s to 4s.

[0085] In some embodiments, the ultrasonic fragmentation conditions in step S2 are: power 195W to 325W, time 5min to 7min, ultrasonic 1s to 3s, pause 2s to 4s.

[0086] In some embodiments, the ultrasonic dispersion conditions in step S2 are: power 160W to 400W, time 10min to 30min; preferably 200W to 400W, time 10min to 30min.

[0087] In some embodiments, the number of membrane passes in step S2 is 11 to 21.

[0088] The present invention will be further described in detail below with reference to specific embodiments.

[0089] In the following embodiments, IF-CLipos represents the nanomedicine delivery system of the present invention, ITA-HpLipos represents liposomes encapsulating itaconic acid (ITA), and Fen-PDLipos represents liposomes encapsulating fenofibrate (Fen).

[0090] In the following embodiments, the frequency range of the ultrasonic instrument used is 20KHz~25KHz, and the maximum ultrasonic power is 650W. Power a%W represents a% of 650W. For example, 30%W is 650×30%W, which is 195W.

[0091] Example 1: Synthesis and Characterization of Histidine-Modified Octadecylamine (His-ODA) and Poly(2-methacryloyloxyethylphosphorylcholine)-Modified Octadecylamine (pMPC-ODA)

[0092] (1) Synthesis and characterization of pMPC-ODA: 1.51 g of ODA was dispersed in 100 mL of chloroform with 1.4 mL of triethylamine at room temperature for 30 min. Then, 0.55 mL of 2-bromoisobutyryl bromide was added dropwise to the mixture, which gradually turned into a transparent solution. The mixture was stirred at 40 °C for 12 h. The solution was washed three times each with 100 mL of 1 M hydrochloric acid solution and ultrapure water. After completely removing chloroform using a rotary evaporator, a white intermediate powder was obtained. 0.41 g of the intermediate product and 1.94 g of MPC were placed together in a double-necked flask, connected to a condenser, and sealed with a rubber stopper, tape, and sealing film. The flask was repeatedly evacuated and nitrogen gas was introduced to remove oxygen. 8 mL of dichloromethane, 8 mL of ethanol, and 600 μL of pentamethyldiethylenetriamine were added using a syringe. Nitrogen gas was continued to be introduced for 30 min, and then 0.21 g of CuBr was added in the dark. The solution was stirred at 40 °C overnight in the dark. The resulting solutions were dialyzed against ethanol and water for 48 h each, and then freeze-dried to obtain pMPC-ODA. The obtained pMPC-ODA was dissolved in a 1:1 mixture of deuterated chloroform and deuterated methanol as a solvent, and its structure was analyzed and characterized by 1H NMR spectroscopy using TMS as an internal standard.

[0093] (2) Synthesis and characterization of His-ODA: Weigh 1 g Boc-His(Boc)-OH, 0.66 g ODA, 1.39 g HATU and 0.47 g DIPEA, dissolve them in 10 mL N,N'-dimethylformamide at room temperature, and stir for 2 h. After the reaction is complete, add 100 mL ultrapure water and mix well, continue stirring for 30 min. Collect the solid precipitate by vacuum filtration and dry it in an oven at 40 °C overnight. Dissolve the obtained intermediate product in 1 mL dichloromethane, then separate by column chromatography (petroleum ether: ethyl acetate = 2:1), remove the remaining organic solvent by rotary evaporation to obtain Boc-His(Boc)-ODA. Add all the obtained Boc-His(Boc)-ODA to 3 mL dioxane hydrochloride and stir at room temperature for 30 min. After the reaction is complete, add 50 mL ultrapure water, and then adjust the pH of the reaction mixture to 8 with 5% NaHCO3 solution while stirring. After stirring for 1 hour, the mixture was vacuum filtered, and the collected solid was dried overnight in an oven at 40°C to obtain His-ODA. The obtained His-ODA was dissolved in a 1:1 mixture of deuterated chloroform and tritated methanol as a solvent, and its structure was analyzed and characterized by 1H NMR spectroscopy using TMS as an internal standard.

[0094] (3) Results of proton nuclear magnetic resonance spectroscopy (NMR) Figure 1 The display shows that in pMPC-ODA... 1 In the H-NMR spectrum, the b peak at chemical shift δ = 0.90 ppm represents the methyl proton signal (3H, CH3-CR-CH2-) at the hydrophobic tail of ODA, the c peak at δ = 1.62 ppm represents the methylene proton signal (2H, -CH2-CR-CH3) at the junction of pMPC and ODA via atom transfer radical polymerization, and the d peak at δ = 3.35 ppm represents the methyl proton signal (2H, -CH2-CR-CH3) at the tertiary amine tail of pMPC, indicating the successful preparation of pMPC-ODA.

[0095] When separating and purifying the final product pMPC-ODA, a 1500MWCO dialysis bag was selected for dialysis. Since the molecular weight of ODA is 270 and the molecular weight of MPC is 295, polymers with a degree of polymerization lower than 4 (with a molecular weight of about 1450) were filtered out. Therefore, the degree of polymerization of the final product is ≥4.

[0096] In the proton NMR spectrum, integral analysis of each characteristic peak yields the number of hydrogen atoms in each peak. By selecting the characteristic peaks belonging to ODA and MPC respectively and comparing them with their original hydrogen atom counts, the number of ODA and pMPC atoms in a synthesized pMPC-ODA can be calculated. The calculated average degree of polymerization of the final product is 5.

[0097] like Figure 2As shown, in His-ODA 1 In the H-NMR spectrum, the b peak at chemical shift δ = 0.90 ppm represents the methyl proton signal (3H, CH3-CR-CH2-) of the hydrophobic tail of ODA, the f peak at δ = 3.16 ppm represents the methylene proton signal (2H, -CH2-NH-CO-) next to the newly formed amide group on ODA, and the h peak at δ = 6.86 ppm represents the olefin hydrogen signal (1H, CH=C) on the His imidazole ring, indicating the successful preparation of His-ODA.

[0098] Example 2: Formulation screening, preparation and characterization of ITA-HpLipos

[0099] (1) Accurately weigh 150 mg of lecithin, 50 mg of cholesterol, and 10 mg or 20 mg of pMPC-ODA. Add 15 mL of a mixed solvent of chloroform and methanol in a volume ratio of 1:1 or 2:1 and dissolve thoroughly at 60 °C. Then, remove the organic solvent by vacuum rotary evaporation to form a thin film. Add 6 mL of preheated ultrapure water and hydrate in a water bath at 60 °C for about 1 h. Collect the obtained primary liposomes and optimize the particle size using a cell sonicator (ultrasonic conditions: power 30% W, time 5 min, interval on 2 s off 3 s). Pass the dispersion through 0.8 μm, 0.45 μm, and 0.22 μm filter membranes for further homogenization to obtain pLipos.

[0100] (2) The formulation screening results for pMPC-ODA content and the ratio of chloroform to methanol mixed solvent are shown in Table 1 below. When pLipos was prepared using a mixed solvent with a volume ratio of chloroform to methanol of 1:1, the polydispersity index (PDI) of pLipos was greater than 0.3, the particle size distribution was large, and it showed instability. However, when the volume ratio was adjusted to 2:1, the PDI was less than 0.3, and the particle size distribution was better. Therefore, a mixed solvent with a volume ratio of chloroform to methanol of 2:1 was selected for the formulation of ITA-HpLipos. Since the later experiments required the positively charged pMPC-ODA to interact with the negatively charged LPS on the bacterial surface, thereby promoting the internalization of the pMPC-modified particles by the bacteria, a higher content of 20 mg pMPC-ODA was selected for the formulation of ITA-HpLipos.

[0101] Table 1. Effects of pMPC-ODA content and the ratio of chloroform to methanol mixed solvent on particle size, zeta potential, and PDI.

[0102]

[0103] (3) Accurately weigh 150 mg of lecithin, 50 mg of cholesterol, 20 mg of pMPC-ODA, and 10 mg or 20 mg of His-ODA. Add 5 mL of methanol and 10 mL of chloroform and dissolve them thoroughly at 60 °C. Then, remove the organic solvent by vacuum rotary evaporation to form a thin film. Add 6 mL of preheated ultrapure water and hydrate in a 60 °C water bath for about 1 h. Collect the obtained primary liposomes and optimize their particle size using a cell sonicator (ultrasonic conditions: power 30% W or 50% W, time 5 min, interval on 2 s off 3 s). Pass the dispersion through 0.8 μm, 0.45 μm, and 0.22 μm filter membranes for further homogenization to obtain HpLipos.

[0104] (4) The results of the formulation screening based on His-ODA content and ultrasonic power of the cell disruptor are shown in Table 2 below. When HpLipos were prepared using ultrasonic power of 30%W, the PDI of HpLipos was greater than 0.3, the particle size distribution was large, and it was unstable. However, when the ultrasonic power was adjusted to 50%W, the PDI of the formulation with His-ODA content of 10mg was less than 0.3, the particle size distribution was better, and the liposomes were relatively stable. Therefore, the formulation with His-ODA content of 10mg and ultrasonic power of 50%W was selected for ITA-HpLipos.

[0105] Table 2. Effects of His-ODA content and ultrasonic power of the cell disruptor on particle size, zeta potential, and PDI.

[0106]

[0107] (5) Preparation and characterization of ITA-HpLipos: Prepared via thin-film dispersion. Accurately weigh 150 mg lecithin, 50 mg cholesterol, 20 mg pMPC-ODA, 10 mg His-ODA, and 500 μg streptavidin. Add 5 mL methanol and 10 mL chloroform and dissolve thoroughly at 60 °C. Then, remove the organic solvent by vacuum rotary evaporation to form a thin film. Add 6 mL of preheated ultrapure water and hydrate in a 60 °C water bath for approximately 1 h. Collect the obtained primary liposomes and optimize their particle size using a cell sonicator (ultrasonic conditions: 50% W power, 5 min time, interval 2 s on, 3 s off). Further homogenize the dispersion by passing it through 0.8 μm, 0.45 μm, and 0.22 μm filters to obtain HpLipos (total volume 6 mL). pLipos does not contain His-ODA; the remaining preparation methods are the same as for HpLipos. The Lipos component contained only 150 mg of lecithin and 50 mg of cholesterol, and the rest of the preparation method was the same as that of HpLipos. ITA loaded with the drug was prepared using a pH gradient method. A 15 mg / mL ITA solution was prepared in 1% glycine hydrochloride buffer at pH 3.3, and then slowly added dropwise to HpLipos while stirring. The volume ratio of ITA solution to HpLipos was 1:2 (i.e., 3 mL of 15 mg / mL ITA solution was added to 6 mL of HpLipos). After the addition was complete, the mixture was reacted in a 50°C water bath for 10 min, and then the reaction was terminated by an ice bath to obtain ITA-HpLipos. Particle size and potential were measured using a Malvern particle size analyzer for formulation characterization.

[0108] (6) Particle size and potential results are as follows Figure 3 As shown, the particle size of ITA-HpLipos is 67.7±0.65 nm. Since there is no His-ODA, the Zeta potentials of Lipos and pLipos are -26.9±1.85 mV and -20.1±1.45 mV, respectively. When His-ODA is modified, the imidazole group can adsorb protons, so the Zeta potentials of HpLipos and ITA-HpLipos change from negative to positive, and are 8.9±1.80 mV and 23.2±1.33 mV, respectively.

[0109] Example 3: Formulation screening, preparation and characterization of Fen-PDLipos

[0110] (1) Accurately weigh 75 mg of lecithin, 25 mg of cholesterol, and 5 mg or 10 mg of dioleoylphosphatidylethanolamine (DOPE). Add 20 μL of oleic acid and 15 mL of chloroform to a 60 μm bath and dissolve thoroughly. Then, remove the organic solvent by vacuum rotary evaporation to form a thin film. Add 6 mL of preheated pH 7.4 PBS buffer and hydrate in a 60 μm water bath for about 1 h. Collect the obtained primary liposomes and optimize the particle size using a cell sonicator (sonication conditions: power 30% W, time 5 min or 7 min, interval on 2 s off 3 s). Pass the dispersion through 0.8 μm, 0.45 μm, and 0.22 μm filters for further homogenization to obtain DLipos.

[0111] (2) The formulation screening results for DOPE content and cell disruption sonication time are shown in Table 3 below. When the cell disruption instrument was used for sonication for 7 min, the polydispersity index (PDI) of DLipos was greater than 0.3, indicating a large particle size distribution and instability. However, when the sonication time was adjusted to 5 min, the PDI was less than 0.3, indicating a better particle size distribution. Therefore, cell disruption instrument sonication for 5 min was selected for the formulation of Fen-PDLipos. Since the later experiments required DOPE to be protonated in the acidic environment of lysosomes to promote the fusion of liposomes and lysosomal membranes to release the drug, a higher content of 10 mg DOPE was selected for the formulation of Fen-PDLipos.

[0112] Table 3. Effects of DOPE content and cell disruption time on particle size, zeta potential, and PDI.

[0113]

[0114]

[0115] (3) Accurately weigh 75 mg of lecithin and 25 mg of cholesterol, add 15 mL of chloroform and dissolve thoroughly in 60 μm solution. Then, remove the organic solvent by vacuum rotary evaporation to form a thin film. Add 6 mL of preheated hydration medium (pH 7.4 PBS buffer) and hydrate in a 60 μm water bath for about 1 h. Collect the obtained primary liposomes. Optimize the particle size using a cell sonicator (ultrasonic conditions: power 30% W, time 5 min, interval 0-2 s-off 3 s). Further homogenize the dispersion by passing it through 0.8 μm, 0.45 μm, and 0.22 μm filters to obtain Lipo. To screen the hybridization conditions for platelet membrane hybrid liposomes, 1 mL of Lipo was mixed with 100 μL of platelet membrane solution extracted and purified from fresh mouse blood, and the mixture was sonicated in a water bath at (160W / 200W / 400W) for 20 min. The mixture, whether sonicated or untreated, was then repeatedly injected through a 0.22 μm polycarbonate filter membrane 11 or 21 times using a liposome extruder to obtain platelet membrane hybrid liposomes.

[0116] (4) The results of the hybrid formulation screening are shown in Table 4 below. When the liposome and platelet membrane mixture was not pretreated with water bath sonication, the polydispersity index (PDI) of the liposomes was greater than 0.3, indicating a large particle size distribution and instability. After water bath sonication pretreatment at 160W and 200W power, the formulation with 11 passes through the membrane had a PDI greater than 0.3, while the formulation with 21 passes through the membrane had a PDI less than 0.3, indicating better stability. For the 400W water bath sonication condition, the PDI of 11 passes through the membrane was better than that of 21 passes. Based on the comparison of several formulations, the formulation with the lowest average PDI was selected, namely the hybrid condition of pretreatment of the liposome and platelet membrane mixture with 200W water bath sonication followed by 21 passes through a 0.22μm polycarbonate membrane, for the formulation of Fen-PDLipos.

[0117] Table 4. Effects of water bath ultrasonic power and extrusion membrane pass count on hybrid liposome particle size and PDI.

[0118] Ultrasonic power (W) Times Size (nm) PDI 11 383.13±10.00 0.44±0.02 21 315.97±6.74 0.32±0.02 160 11 288.10±7.37 0.31±0.01 160 21 262.23±2.40 0.25±0.02 200 11 306.03±8.03 0.37±0.01 200 21 127.60±6.32 0.21±0.01 400 11 279.83±6.01 0.23±0.02 400 21 246.03±4.91 0.31±0.01

[0119] (5) Flow cytometry was used to investigate the differences in neutrophil uptake of liposomes with different platelet membrane hybridization ratios, thereby screening for suitable hybridization ratios. 75 mg of lecithin and 25 mg of cholesterol were accurately weighed and dissolved in 15 mL of chloroform at 60 μm. The organic solvent was then removed by vacuum rotary evaporation to form a thin film. 6 mL of preheated hydration medium (pH 7.4 PBS buffer) was added, and the mixture was hydrated in a 60 μm water bath for approximately 1 h. The resulting primary liposomes were collected and their particle size optimized using a cell sonicator (ultrasonic conditions: 30% W, 5 min, interval 0-2 s-3 s-off). The dispersion was further homogenized by passing it through 0.8 μm, 0.45 μm, and 0.22 μm filters to obtain Lipo. Platelet membrane hybrid liposomes PLipos were prepared according to phospholipid-to-platelet membrane protein ratios of 1:500, 1:250, 1:100, and 1:50. After measuring the platelet membrane protein concentration, the solutions were diluted to 100 μL according to the groupings, and the platelet membrane solutions were sonicated in a 200W water bath for 20 min. 1 mL of Lipo was mixed with each concentration of platelet membrane solution, and the mixture was repeatedly injected through a 0.22 μm polycarbonate filter membrane 21 times using a liposome extruder to obtain PLipos. After preparing Lipo and PLipos with phospholipid-to-platelet membrane protein ratios of 1:500, 1:250, 1:100, and 1:50, they were incubated with 10 mg / mL DiR dye under light-protected conditions for 40 min. Neutrophils were seeded at a density of 3 × 10⁵ cells / well in 24-well plates. Stained Lipo and PLipos were added to each well in the dark (final DiR concentration of 50 μg / mL and final phospholipid concentration of 256 μg / mL). After incubation at 37°C for 1 h, the cells were washed twice with HBSS and resuspended in 500 μL of HBSS. The fluorescence intensity of DiR in neutrophils of each group was detected by flow cytometry.

[0120] (6) Screening results of platelet membrane hybridization ratio as follows Figure 4 As shown, at an uptake time of 1 hour, the fluorescence intensity of PLipos with different hybridization ratios in neutrophils was significantly higher than that in the Lipo group. Furthermore, the fluorescence intensity within neutrophils gradually increased with increasing hybridization ratio, indicating that the presence of the platelet membrane promoted neutrophil uptake of liposomes. The neutrophil uptake of PLipos with hybridization ratios of 1:50 and 1:100 was significantly higher than that of other PLipos. Since there was no significant difference in neutrophil uptake between 1:50 and 1:100 PLipos, a hybridization ratio of 1:100 was chosen for the Fen-PDLipos formulation to reduce the amount of platelet membrane used.

[0121] (7) Preparation and characterization of Fen-PDLipos: Prepared by thin-film dispersion method. Accurately weigh 75 mg lecithin, 25 mg cholesterol, 10 mg DOPE, 24 mg Fen, and 1 mg biotin. Add 20 μL oleic acid and 15 mL chloroform and dissolve thoroughly at 60 °C. Then, remove the organic solvent by vacuum rotary evaporation to form a thin film. Add 6 mL of preheated pH 7.4 PBS buffer and hydrate in a 60 °C water bath for about 1 h. Collect the obtained primary liposomes and optimize the particle size using a cell sonicator (sonication conditions: power 30% W, time 5 min, interval 0-2 s-off 3 s). The dispersion is further homogenized by passing it through 0.8 μm, 0.45 μm, and 0.22 μm filters to obtain DOPE-modified liposomes loaded with Fen (total volume 6 mL). After measuring the platelet membrane protein concentration, the platelet membrane is diluted to 100 μL and sonicated in a 200 W water bath for 20 min. Six mL of DOPE-modified liposomes loaded with Fen were mixed with 100 μL of platelet membrane solution. The mixture was repeatedly injected through a 0.22 μm polycarbonate filter membrane 21 times using a liposome extruder to obtain Fen-PDLipos. PDLipos were prepared without Fen loading, using the same method as Fen-PDLipos. Particle size and potential were measured using a Malvern particle size analyzer for formulation characterization.

[0122] (8) Particle size and potential results are as follows Figure 5 As shown, the particle size of Fen-PDLipos is 124.3±0.57nm; the Zeta potentials of Lipo, PLipos, PDLipos and Fen-PDLipos are similar, ranging from -10mV to -20mV.

[0123] Example 4: Preparation and Characterization of Companion Liposomes IF-CLipos

[0124] (1) Preparation of IF-CLipos: IF-CLipos are formed by linking ITA-HpLipos and Fen-PDLipos through the interaction of avidin and biotin. The ITA-HpLipos prepared in Example 2 and the Fen-PDLipos prepared in Example 3 were mixed at a volume ratio of 1:1. In this example, 6 mL of the ITA-HpLipos prepared in Example 2 and 6 mL of the Fen-PDLipos prepared in Example 3 were mixed and incubated at 37°C for 30 min to link them.

[0125] (2) Morphological characterization of IF-CLipos: First, the morphology of IF-CLipos was examined by transmission electron microscopy. After preparing IF-CLipos, 10 μL was dropped onto a copper grid and allowed to stand for 1 min to dry. Then, 3% phosphotungstic acid negative staining solution was dropped onto the copper grid and stained for 20 s. The liquid was absorbed with filter paper, and the morphology was observed under a transmission electron microscope with a working voltage of 120 kV. To examine the morphology of IF-CLipos by laser confocal microscopy, DiD-HpLipos and DiI-PDLipos were prepared, with DiD and DiI concentrations of 16.7 μg / mL. The two liposomes were mixed at a volume ratio of 1:1 and incubated at 37 °C for 30 min to ligate them. A clean glass slide was taken, and 5 μL of anti-fluorescence quenching mounting solution and 5 μL of DiD and DiI labeled IF-CLipos were added. After covering with a coverslip, the slide was observed under a laser confocal microscope at 100x magnification.

[0126] (3) The morphological characterization results of IF-CLipos are as follows: Figure 6 As shown, ITA-HpLipos and Fen-PDLipos are linked together through the non-covalent interaction of avidin and biotin to form IF-CLipos, and both have typical liposome phospholipid bilayer structures with regular shapes, linked together in a "hand-in-hand" manner. Figure 7 This is a representative fluorescence image obtained by laser confocal microscopy of IF-CLipos.

[0127] Example 5: Investigation of IF-CLipos' in vivo neutrophil hijacking function

[0128] (1) The uptake of PDLipos, HpLipos, and CLipos by circulating neutrophils in mice was investigated by flow cytometry to evaluate the hijacking ability of IF-CLipos on neutrophils. Cou6-HpLipos and DiR-PDLipos were prepared according to the steps described above and linked to form CLipos, with the concentrations of Cou6 and DiR both at 200 μg / mL. BALB / c mice were randomly divided into a blank group, a DiR-PDLipos group, a Cou6-HpLipos group, and a CLipos group. All mice were injected intravenously with 100 μL of PE-Ly6G at a concentration of 50 μg / mL to label circulating neutrophils. Subsequently, except for the blank group, each group of mice was injected intravenously with 100 μL of the corresponding liposome. After mice were protected from light for 1 hour, fresh blood was collected and neutrophils were extracted. The fluorescence intensity of DiR and Cou6 in the neutrophils of each group of mice was detected by flow cytometry.

[0129] (2) Experimental results Figure 8The results showed that PDLipos, due to its membrane hybridization and inclusion of a platelet membrane, could be recognized and phagocytosed by neutrophils in the systemic circulation, with an uptake rate of 16.61 ± 0.60%. In contrast, for HpLipos, which lacked platelet membrane hybridization, the uptake rate by neutrophils was only 0.25 ± 0.13%. When PDLipos and HpLipos linked to form CLipos, HpLipos could hijack neutrophils via PDLipos, resulting in a neutrophil uptake rate of 7.63 ± 0.60%. These results indicate that IF-CLipos can hijack neutrophils, with Fen-PDLipos recognizing and binding to neutrophils through the platelet membrane, and ITA-HpLipos being co-uptaken by neutrophils along with Fen-PDLipos through the interaction of biotin and avidin.

[0130] Example 6: Determination of particle size and potential changes of IF-CLipos at different pH values

[0131] (1) ITA-HpLipos and Fen-PDLipos were prepared according to the methods described in Examples 2 and 3. 100 μL of each liposome was added to 900 μL of PBS buffer with pH 7.4, 6.5, 5.5 and 4.5 respectively. After standing for 1 h, 1 mL was transferred to a particle size cuvette or 700 μL was transferred to a potentiometer. The particle size and potential were measured using a Malvern nanoparticle size analyzer.

[0132] (2) Experimental results Figure 9 The results showed that with increasing acidity, the particle size of ITA-HpLipos remained almost unchanged, while the Zeta potential changed from negative to positive and gradually increased. This is because His-ODA has a strong nucleophilic imidazole ring, which can be continuously protonated in an acidic environment, making the surface of ITA-HpLipos positively charged. However, when the pH value of Fen-PDLipos decreased to 5.5 and 4.5, i.e., in a simulated lysosomal environment, the particle size of Fen-PDLipos suddenly increased, the liposome state became unstable, and the absolute value of the Zeta potential decreased. This is because the DOPE on Fen-PDLipos protonated at the hydroxyl end in an acidic environment, neutralizing the original negative charge carried by the liposome, and the DOPE formed a hexagonal crystal phase, thus causing liposome membrane fusion. These results indicate that ITA-HpLipos can adsorb H+ through His-ODA. + Increasing the pH within the lysosome, followed by H + With Cl - A large amount of water is pumped into the lysosome, causing the lysosome to rupture and escape. At the same time, ITA-HpLipos itself remains stable and is not destroyed. Meanwhile, DOPE in Fen-PDLipos is protonated in an acidic environment and triggers the fusion of the liposome membrane and the lysosome membrane, thereby releasing the drug fenofibrate.

[0133] Example 7: Investigation of the neutrophil educational function of IF-CLipos

[0134] (1) The effect of Fen-PDLipos in IF-CLipos on the expression of CXCR2 receptor protein in neutrophils was investigated by immunofluorescence staining. Neutrophils were extracted from mouse bone marrow and subjected to immunofluorescence staining at a concentration of 8 × 10⁻⁶ cells / mL. 5 Cells were seeded at a density of 10 μg / mL into 24-well plates containing poly-L-lysine smears. Except for the control group, LPS was added to each group to a final concentration of 10 μg / mL. Free fenofibrate solution was prepared with DMSO. Fen-PDLipos was prepared according to the method described above. Fenofibrate and Fen-PDLipos were administered to each well at a final concentration of 40 μg / mL. After administration, the cells were incubated at 37°C for 1 h.

[0135] (2) After incubation, remove the culture medium, add 4% paraformaldehyde for fixation, place on ice for 10 min, then at room temperature for 20 min. Weigh 0.5 g BSA and dissolve in 50 mL PBS buffer, then add 50 μL Tween 20 and vortex to dissolve, thus obtaining 1% BSA blocking solution. After fixation, aspirate the liquid and wash three times with PBS, add 500 μL of blocking solution, and incubate at 37 °C for 30 min.

[0136] (3) Discard the blocking solution, incubate with a 100-fold diluted anti-CXCR2 antibody at 4°C overnight. After recovering the remaining anti-CXCR2 antibody, add 500 μL of blocking solution and incubate at 37°C for 10 min. Discard the blocking solution, incubate with a 500-fold diluted Cy3-modified goat anti-rabbit IgG antibody at 37°C for 1 h. Add 300 μL of 30 μg / mL DAPI to stain the cell nuclei and incubate at room temperature for 10 min. Take a clean glass slide, add 5 μL of anti-fluorescence quenching mounting solution, pick up the slide, and place the cell surface in contact with the mounting medium. Observe under a laser confocal microscope at 100x magnification.

[0137] (4) Experimental results Figure 10 The results showed that after stimulation with 10 μg / mL LPS for 1 h, the expression of CXCR2 on the surface of neutrophils was downregulated due to internalization. Compared with free Fen, Fen-PDLipos was more easily taken up by neutrophils and could significantly inhibit the internalization of CXCR2 receptor protein on the surface of neutrophils, restoring its expression level to a level close to that of normal neutrophils.

[0138] Example 8: Neutrophils clear extracellular bacteria by releasing ITA-HpLipos via NETs

[0139] (1) The antibacterial effect of IF-CLipos on enhancing NETs was investigated by measuring the growth curves of Escherichia coli and Pseudomonas aeruginosa within 48 h. IF-CLipos was prepared and diluted to an itaconic acid concentration of 2560 μg / mL for later use. Mouse bone marrow neutrophils were extracted and the cell density was adjusted to 2 × 10⁻⁶ cells / mL. 5 = / mL, add PMA to a final concentration of 100nM and incubate at 37℃ in the dark for 4h to induce NETs formation. Take another neutrophil suspension, add an equal volume of IF-Clipos with ITA concentration of 2560μg / mL and incubate for 2h, then add PMA to a final concentration of 100nM and incubate at 37℃ in the dark for 4h.

[0140] (2) Both E. coli and P. aeruginosa bacterial cultures were diluted to 1×10⁻⁶. 5 CFU / mL, 100 μL per well in a 96-well plate. The Control group received 100 μL of LB liquid medium per well; the NETs group received 50 μL of neutrophil NETs suspension and 50 μL of LB liquid medium per well; the IF-CLipos group received 50 μL of IF-CLipos and 50 μL of LB liquid medium per well; and the NETs+IF-CLipos group received 100 μL of neutrophil NETs suspension containing IF-CLipos per well (final itaconic acid concentration 640 μg / mL, neutrophil to bacterial colony ratio 1:1).

[0141] (3) The 96-well plate was placed in a bacterial incubator at 37℃. At 0h, 4h, 8h, 12h, 24h, 28h, 32h, 36h, and 48h of incubation, the absorbance of each well was measured at a wavelength of 600nm using a microplate reader. The growth fold of E. coli and P. aeruginosa at each time point was calculated using the following formula. OD600(t0) is the absorbance value of each well at 600nm at 0h, and OD600(tn) is the absorbance value of each well at 600nm at each time point.

[0142] Bacteria growth rate = OD 600 (t n ) / OD 600 (t0)

[0143] (4) Experimental results Figure 11The results showed that the growth rate of *E. coli* in the control group was 31.2 ± 3.2 times at 48 h, while that of *P. aeruginosa* was 40.8 ± 2.0 times. IF-CLipos enhanced the inhibitory effect of NETs on bacterial growth. After administration of NETs + IF-CLipos, the growth rate of *E. coli* in the NETs + IF-CLipos group was 4.7 ± 1.8 times at 48 h, and that of *P. aeruginosa* was 10.1 ± 1.0 times at 48 h, significantly lower than the bacterial growth rate in the control group, and superior to the effect of NETs and IF-CLipos alone. This indicates that compared with NETs released from neutrophils, the extracellular antibacterial effect of NETs released after incubation with neutrophils by IF-CLipos of this invention is significantly enhanced, and superior to the extracellular antibacterial effect of IF-CLipos itself.

[0144] Example 9: Neutrophils clear intracellular bacteria by releasing ITA-HpLipos via NETs.

[0145] (1) Prepare IF-CLipos according to the method described in Example 4, and dilute itaconic acid to a concentration of 1280 μg / mL for later use. Extract mouse bone marrow neutrophils and adjust the cell density to 1 × 10⁻⁶ cells / mL. 7 Add 100 nM PMA to 500 μL of neutrophil suspension and incubate at 37°C in the dark for 4 h. Separately, take 500 μL of neutrophil suspension, add 500 μL of IF-Clipos with ITA concentration of 1280 μg / mL and incubate for 2 h, then add 100 nM PMA and incubate at 37°C in the dark for 4 h to induce NETs formation.

[0146] (2) RAW264.7 macrophages were stored at a density of 5×10⁻⁶ cells / cm². 5 / wells were inoculated into 24-well plates containing standard cell crawling sheets and incubated overnight at 37°C and 5% CO2. After obtaining the S. typhimurium bacterial culture, the culture was diluted to 1×10⁻⁶ with DMEM medium. 7 CFU / mL, add 500 μL of bacterial culture to a 24-well plate and co-incubate with RAW264.7 macrophages for 1 h. At this point, the ratio of bacterial colonies to macrophages is 10:1. After incubation, discard the supernatant and add DMEM medium containing 100 μg / mL gentamicin for 1 h to eliminate extracellular bacteria. Discard the supernatant and wash three times with PBS to obtain the Salmonella typhimurium invasion model of intracellular bacteria in macrophages.

[0147] (3) Aspirate the supernatant from the RAW264.7 macrophage culture and administer the drugs according to the groupings. Add 1 mL of complete DMEM medium to each well in the Control group; add 500 μL of neutrophil NETs suspension and 500 μL of complete DMEM medium to each well in the NETs group; add 500 μL of IF-CLipos and 500 μL of complete DMEM medium (itaconic acid final concentration 640 μg / mL) to each well in the NETs+IF-CLipos group; add 1 mL of neutrophil NETs suspension containing IF-CLipos to each well. At this point, the ratio of neutrophil count to bacterial colony count to macrophage count is 10:10:1. After drug administration, incubate the cells at 37℃ and 5% CO2 for 12 h.

[0148] (4) Discard the culture supernatant, wash three times with PBS, and add 250 μL of 0.1% Triton X-100 solution (10 μL Triton X-100 added to 10 mL PBS) to each well to lyse the cells for 2 h. Take 5 μL of cell lysate from each group, dilute it to 100 μL with LB liquid medium, drop it onto LB solid medium, and spread it evenly with a disposable spreader until the liquid evaporates. Incubate in a bacterial incubator at 37℃ for 12 h, and count the number of colonies that grow on the plates.

[0149] (5) Experimental results Figure 12 The results showed that the NETs+IF-CLipos group had a superior antibacterial effect compared to NETs alone. The macrophage lysate containing intracellular bacteria showed almost no colony growth on agar plates, and the number of residual S. typhimurium intracellularly was less than 1 after quantifying the colony count per macrophage. This indicates that compared to NETs released from neutrophils, the intracellular antibacterial effect of NETs released after incubation with IF-CLipos of this invention is significantly enhanced, and superior to the intracellular antibacterial effect of IF-CLipos itself.

[0150] Example 10

[0151] An acute intestinal infection model was established in mice by gavage administration of Salmonella Typhimurium. BALB / c mice were randomly divided into a healthy group, a model group, a Fen-PDLipos treatment group, and an IF-CLipos treatment group. After fasting and depriving mice of water for 6 hours, they were first gavaged with 100 μL of 5% NaHCO3 solution to neutralize gastric acid. 30 minutes later, healthy mice were gavaged with 400 μL of physiological saline, while mice in the model and treatment groups were gavaged with 400 μL of 1×10⁻⁶ saline solution. 9CFU / mL Salmonella Typhimurium bacterial suspension. Fen-PDLipos and IF-CLipos were prepared according to the methods in Examples 3 and 4. Fen-PDLipos and IF-CLipos were administered via tail vein injection according to the grouping and Fen dosage of 25 mg / kg. Forty-eight hours after modeling and drug administration, mice were euthanized by cervical dislocation and dissected. Appropriate lengths of the small intestine and jejunum were selected, fixed with 4% paraformaldehyde, graded dehydration, embedded in paraffin, sectioned, stained with Ly6G fluorescent antibody, and observed under 10x magnification in an integrated cell imaging system.

[0152] Experimental results ( Figure 13 The results showed that, compared with the Model group, treatment with Fen-PDLipos and IF-CLipos significantly increased the green fluorescence signal of neutrophils at the site of intestinal infection in mice. This indicates that treatment "trains" neutrophils to restore their chemotaxis towards the infection site, thereby increasing neutrophil infiltration and promoting pathogen clearance. Furthermore, IF-CLipos was more effective than Fen-PDLipos in restoring neutrophil chemotaxis towards the infection site, exhibiting a stronger green fluorescence signal.

[0153] Example 11

[0154] A mouse model of acute intestinal infection was established and mice were grouped according to the method in Example 10. Drugs were administered via tail vein injection according to the grouping. Mice were euthanized by cervical dislocation and dissected. Appropriate lengths of small intestine and jejunum were selected, fixed with 4% paraformaldehyde, dehydrated in a gradient manner, embedded in paraffin, sectioned, and rapidly Gram-stained. The bacterial count was observed under 10x magnification using an integrated cell imaging system.

[0155] Experimental results ( Figure 14 The results showed that in the Model group, due to the presence of a large number of bacteria in the intestinal infection, Fen-PDLipos treatment, while providing some antibacterial protection by increasing neutrophil infiltration at the infection site, still left some bacteria behind. In contrast, after IF-CLipos treatment, almost no pathogens remained in the mouse intestines, indicating that the therapeutic effect of IF-CLipos was significantly superior to Fen-PDLipos. This may be because after IF-CLipos trains neutrophils to chemotactically move to the intestinal infection site, the neutrophils are stimulated by the pathogens to form NETs containing ITA-HpLipos. The ITA-HpLipos are then taken up by the pathogens and release itaconic acid, thereby enhancing the pathogen-clearing effect of the NETs.

[0156] Example 12

[0157] A mouse model of acute intestinal infection was established according to the method in Example 10. BALB / c mice were randomly divided into a healthy group, a model group, a Fen-PDLipos combined with ITA-HpLipos treatment group, and an IF-CLipos treatment group. ITA-HpLipos and Fen-PDLipos were prepared according to the methods in Examples 2 and 3, but without the addition of streptavidin and biotin. IF-CLipos were prepared according to the method in Example 4. The mice were administered the medication via tail vein injection according to their respective groups. In the Fen-PDLipos combined with ITA-HpLipos treatment group, Fen-PDLipos was injected via tail vein at a dose of 25 mg / kg, and ITA-HpLipos was injected via tail vein at a dose of 30 mg / kg. In the IF-CLipos treatment group, Fen-PDLipos was injected at a dose of 25 mg / kg, and ITA-HpLipos at a dose of 30 mg / kg. Mice were euthanized by cervical dislocation and dissected. A suitable length of small intestine and jejunum segment was selected, fixed with 4% paraformaldehyde, dehydrated in a gradient manner, embedded in paraffin, sectioned, and rapidly Gram stained. The amount of bacteria was observed under 10x magnification using an integrated cell imaging system.

[0158] The experimental results showed that the intestinal tract of the Model group contained a large number of bacteria. Both the Fen-PDLipos combined with ITA-HpLipos treatment group and the IF-CLipos treatment group showed significant therapeutic effects. However, a small amount of bacteria remained in the intestinal tract of mice in the Fen-PDLipos combined with ITA-HpLipos treatment group, while almost no bacteria remained in the intestinal tract of mice in the IF-CLipos treatment group. This indicates that the IF-CLipos treatment group was more effective than the Fen-PDLipos combined with ITA-HpLipos treatment group. This may be because in the IF-CLipos treatment group, ITA-HpLipos can be taken up by neutrophils along with Fen-PDLipos through the streptavidin-biotin linkage, and then driven to the infection site by neutrophils, exerting its effect through the NETs released by neutrophils, thus enhancing the anti-infective effect. In contrast, in the Fen-PDLipos combined with ITA-HpLipos treatment group, ITA-HpLipos was difficult to be taken up by neutrophils, and could not be driven to the infection site or exert its effect through the released NETs.

[0159] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for preparing a nanomedicine delivery system targeting neutrophils, characterized in that, The following steps are involved: S1. Lecithin, cholesterol, poly(2-methacryloyloxyethylphosphorylcholine) modified fatty amine, histidine modified fatty amine, ligand or receptor are added to organic solvent 1 to prepare liposome 1; while stirring, a solution containing drug 1 is dropped into the liposome 1 to obtain liposome 1 encapsulated with drug 1; the drug 1 is an anti-inflammatory drug. S2. Lecithin, cholesterol, dioleoylphosphatidylethanolamine, drug 2, and a receptor or ligand corresponding to the ligand or receptor added in step S1 are added to organic solvent 2 to prepare liposome 2; the liposome 2 is mixed with platelet membrane to prepare liposome 2 encapsulated with drug 2; the drug 2 is a drug that induces neutrophils to chemotaxis toward the site of infection. S3. The liposome 1 encapsulating drug 1 and the liposome 2 encapsulating drug 2 are mixed and incubated, and the corresponding ligands and receptors in the liposome 1 and liposome 2 are connected to obtain the drug 1. The ligand and receptor are biotin and streptavidin, respectively. In step S1, streptavidin is added, and in step S2, biotin is added. In step S1, the mass ratio of lecithin to streptavidin is (150~200):(0.5~3). In step S2, the mass ratio of lecithin to biotin is (75~100):(1~5); The drug 1 is selected from at least one of itaconic acid and antibiotics; The drug 2 is selected from at least one of GRK2 inhibitors and ERK inhibitors.

2. The preparation method according to claim 1, characterized in that, Drug 1 is itaconic acid, and drug 2 is fenofibrate; In step S1, the mass ratio of lecithin to itaconic acid is (150~200):(9~45); and / or, In step S2, the mass ratio of lecithin to fenofibrate is (75~100):(20~30).

3. The preparation method according to claim 1 or 2, characterized in that, In step S3, the liposomes 1 encapsulating drug 1 and liposomes 2 encapsulating drug 2 are mixed and incubated at a mass ratio of drug 1 to drug 2 of (6~30): (20~30).

4. The preparation method according to claim 1, characterized in that, The poly(2-methacryloyloxyethylphosphatidylcholine) modified fatty amine is selected from poly(2-methacryloyloxyethylphosphatidylcholine) modified octadecylamine, poly(2-methacryloyloxyethylphosphatidylcholine) modified stearoylphosphatidylethanolamine, poly(2-methacryloyloxyethylphosphatidylcholine) modified hydrogenated soybean phosphatidylcholine, and poly(2-methacryloyloxyethylphosphatidylcholine) modified dipalmitoylphosphatidylcholine; and / or, The histidine-modified fatty amine is selected from histidine-modified octadecylamine, histidine-modified stearoylphosphatidylethanolamine, histidine-modified hydrogenated soybean phosphatidylcholine, and histidine-modified dipalmitoylphosphatidylcholine.

5. The preparation method according to claim 4, characterized in that, The poly(2-methacryloyloxyethylphosphorylcholine) modified fatty amine is selected from poly(2-methacryloyloxyethylphosphorylcholine) modified octadecylamine, and the histidine modified fatty amine is selected from histidine modified octadecylamine. In step S1, the mass ratio of lecithin, cholesterol, poly(2-methacryloyloxyethylphosphorylcholine)-modified octadecylamine, and histidine-modified octadecylamine is (150~200):50:(10~20):(10~20); and / or, In step S2, the mass ratio of lecithin, cholesterol, and dioleoylphosphatidylethanolamine is (75~100):25:(10~20); and / or, In step S2, the mass ratio of lecithin to total platelet membrane protein is 1:(50~500).

6. The preparation method according to claim 5, characterized in that, In step S2, the mass ratio of lecithin to total platelet membrane protein is 1:(50~250).

7. The preparation method according to claim 6, characterized in that, In step S2, the mass ratio of lecithin to total platelet membrane protein is 1:(50~100).

8. A nanomedicine delivery system targeting neutrophils, characterized in that, It is prepared by the preparation method according to any one of claims 1 to 7.

9. The application of the neutrophil-targeting nanomedicine delivery system as described in claim 8 in the preparation of anti-infective drugs.

10. An anti-infective drug, characterized in that, It includes an active ingredient and a pharmaceutically acceptable carrier; the active ingredient includes the neutrophil-targeting nanomedicine delivery system as described in claim 8.

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