Nano-drug delivery system for targeting neutrophil as well as preparation method and application of nano-drug delivery system

Abstract

The invention relates to a nano-drug delivery system targeting neutrophil and a preparation method and application thereof. The nano-drug delivery system is prepared by the following method: S1, preparing a liposome 1 wrapping a drug 1; s2, preparing a liposome 2 wrapping a medicine 2; and S3, mixing and incubating the liposome 1 wrapping the medicine 1 and the liposome 2 wrapping the medicine 2, and connecting the corresponding ligand and receptor in the liposome 1 and the liposome 2 to obtain the liposome. The ligand and the receptor are respectively biotin and streptavidin. The nano-drug delivery system is obtained by successfully connecting two drug liposomes with different functions through a ligand and a receptor system, so that the chemotaxis of neutrophil to an infected part can be enhanced, the antibacterial property of NETs can be improved, and the nano-drug delivery system has a very good anti-infection effect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a nano drug delivery system targeting neutrophils, and a preparation method and application thereof. Background Art

[0002] Neutrophils can migrate to the site of infection in response to the induction of chemokines, and release extracellular traps (NETs) composed of histones, granules, elastase, myeloperoxidase, DNA, etc. to capture and remove pathogens that invade the body. However, in severe infectious diseases such as sepsis, the continuous stimulation of intracellular and extracellular pathogens will cause the internalization of chemokine receptor CXCR2 on the surface of neutrophils, weakening their chemotactic ability to lesions. In addition, the bactericidal effect of NETs is insufficient. Some pathogens can degrade NETs by forming nucleases, thereby escaping the capture and killing of NETs. It is difficult to meet the anti-infection needs by relying solely on NETs.

[0003] At present, nanomedicines designed based on the natural tropism of neutrophils to the lesion area are widely studied. The use of neutrophils to transport nanomedicines is generally based on two different strategies. The first is to load nanomedicines on extracted neutrophils in vitro. Due to the short life span of neutrophils, complex operations will seriously affect the vitality of neutrophils, and there is a risk of in vitro contamination, so the feasibility of this strategy is low; the second strategy is to allow nanomedicines to hijack neutrophils in the systemic circulation. This strategy circumvents the defects of the first strategy, but requires nanoparticles to have the ability to efficiently bind to neutrophils. In addition, since neutrophils cannot respond to chemokines to migrate to the site of infection during severe infection, pathogens cannot be cleared and spread rapidly, causing the disease to worsen. Therefore, "re-education" measures for the chemotaxis of neutrophil lesions are crucial for the treatment of related diseases. The current main measures are to intervene in the internalization-related signaling pathways of the chemokine receptor CXCR2 on the surface of neutrophils. Related studies have shown that neutrophil NETs can release drugs in the neutrophil cytoplasm to the extracellular space, such as releasing antimicrobial peptides contained in neutrophils themselves to eliminate extracellular pathogens; or NETs are recognized and phagocytosed by macrophages to eliminate intracellular parasites in macrophages.

[0004] At present, it is necessary to develop a drug that can truly restore the chemotaxis of neutrophils at the infection site 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 the present invention is to provide a nano drug delivery system targeting neutrophils, which can both hijack and educate neutrophils to restore their chemotaxis to the infection site, and enhance the effect of neutrophil NETs against intracellular and extracellular bacteria, thereby achieving better therapeutic effects.

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

[0007] S1, adding lecithin, cholesterol, poly (2-methacryloyloxyethyl phosphorylcholine) modified fatty amine, histidine modified fatty amine, ligand or receptor into organic solvent 1 to prepare liposome 1; dropping a solution containing drug 1 into the liposome 1 while stirring to obtain liposome 1 encapsulating drug 1; the drug 1 is an anti-inflammatory drug;

[0008] S2, adding lecithin, cholesterol, dioleoylphosphatidylethanolamine, drug 2, and a receptor or ligand corresponding to the ligand or receptor added in step S1 into an organic solvent 2 to prepare liposome 2; mixing the liposome 2 with a platelet membrane to prepare a liposome 2 encapsulating drug 2; the drug 2 is a drug that induces neutrophil chemotaxis to the infection site;

[0009] S3, mixing and incubating the liposome 1 encapsulating the drug 1 and the liposome 2 encapsulating the drug 2, so that the corresponding ligands and receptors in the liposome 1 and the liposome 2 are connected, and the result is obtained;

[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, 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.

[0015] In some embodiments, the drug 1 is itaconic acid and the 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 liposome 1 encapsulating the drug 1 and the liposome 2 encapsulating the drug 2 are mixed and incubated at a mass ratio of the drug 1 to the drug 2 of (6-30):(20-30).

[0019] In some of the 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 of the 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-methacryloyloxyethylphosphorylcholine)-modified fatty amine is selected from poly(2-methacryloyloxyethylphosphorylcholine)-modified octadecylamine, and the histidine-modified fatty amine is selected from histidine-modified octadecylamine.

[0022] In some of the embodiments, in step S1, the mass ratio of phosphatidylcholine, cholesterol, poly(2-methacryloyloxyethylphosphorylcholine)-modified octadecylamine, and histidine-modified octadecylamine 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] The second aspect of the present invention is to provide a nano drug delivery system targeting neutrophils, which is prepared by the preparation method described above.

[0026] The third aspect of the present invention is to provide the use of the nano drug delivery system targeting neutrophils as described above in the preparation of anti-infective drugs.

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

[0028] The present invention provides a nano drug delivery system targeting neutrophils, and two drug liposomes with different functions (liposome 1 encapsulating drug 1 and liposome 2 encapsulating drug 2) are successfully connected through a ligand and receptor system to obtain the nano drug delivery system. Among them, the liposome 2 encapsulating drug 2 is hybridized with the platelet membrane, so that the nano drug delivery system can be recognized by neutrophils and engulfed into the neutrophils. After the nano drug delivery system is endocytosed into the neutrophils, the liposome 2 encapsulating drug 2 can fuse with the neutrophil lysosomal membrane in the acidic environment of the lysosome, release drug 2, and enhance the chemotaxis of the lesion of neutrophils; and the liposome 1 encapsulating drug 1 can enter the neutrophil cytoplasm in a complete form, be delivered to the infection site by the neutrophils and released through NETs, ​​and enhance the intracellular and extracellular antibacterial effects of NETs. In addition, the release of drug 1 further activates the body's natural immune system, thereby further enhancing the chemotaxis of neutrophils to the infection site.

[0029] The present invention successfully prepares a nano drug delivery system comprising two different functional drug liposomes through a ligand and receptor connection system. In addition, the inventor has found through a large number of experiments and studies that the mass ratio of liposome lecithin to ligand and receptor has a very important influence on the stability of the nano drug delivery system obtained after the two drug liposomes can be efficiently and successfully connected through ligand and receptor, and the appropriate mass ratio of lecithin to ligand and receptor is screened to ensure the successful preparation of the nano drug delivery system and the curative effect in vivo. Further, the ratio of drug 1 and drug 2 has a significant effect on the anti-infective effect of the nano drug delivery system, and the appropriate mass ratio of drug 1 and drug 2 on the nano drug delivery system can simultaneously enhance the chemotaxis and bactericidal performance of neutrophils to the infection site, so that the nano drug delivery system of the present invention has a very good anti-infective effect. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0032] Figure 3 are the particle size and Zeta potential of ITA-HpLipos.

[0033] Figure 4 Screening platelet membrane hybridization ratio results for flow cytometry.

[0034] Figure 5 are the particle size and Zeta potential of Fen-PDLipos.

[0035] Figure 6 Representative transmission electron microscopy images of IF-CLipos.

[0036] Figure 7 Representative fluorescence images of IF-CLipos under confocal laser scanning microscope.

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

[0038] Fig. 9 The particle size and Zeta potential changes of IF-CLipos under different pH conditions.

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

[0040] Fig.11 Neutrophils release ITA-HpLipos through NETs to clear extracellular bacteria.

[0041] Fig.12 Neutrophils release ITA-HpLipos through NETs to clear intracellular bacteria.

[0042] Fig.13 These are the results of neutrophil infiltration detection in the intestinal infection lesions of mice.

[0043] Fig.14 The results of the bacterial content test in the mouse intestine. DETAILED DESCRIPTION

[0044] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0045] The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

[0046] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0047] In addition, as used in the present invention, the term "or" is an inclusive "or" symbol and is equivalent to the term "and / or", unless the context clearly dictates otherwise. The term "based on" is not exclusive and allows for being based on other factors not described, unless the context clearly dictates otherwise. In addition, throughout the specification, the meanings of "a", "an", and "the" include plural referents. The meaning of "in..." includes "in..." and "on...".

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

[0049] S1, adding lecithin, cholesterol, poly (2-methacryloyloxyethyl phosphorylcholine) modified fatty amine, histidine modified fatty amine, ligand or receptor into organic solvent 1 to prepare liposome 1; dropping a solution containing drug 1 into the liposome 1 while stirring to obtain liposome 1 encapsulating drug 1; the drug 1 is an anti-inflammatory drug;

[0050] S2, adding lecithin, cholesterol, dioleoylphosphatidylethanolamine, drug 2, and a receptor or ligand corresponding to the ligand or receptor added in step S1 into an organic solvent 2 to prepare liposome 2; mixing the liposome 2 with a platelet membrane to prepare a liposome 2 encapsulating drug 2; the drug 2 is a drug that induces neutrophil chemotaxis to the infection site;

[0051] S3, mixing and incubating the liposome 1 encapsulating the drug 1 and the liposome 2 encapsulating the drug 2, so that the corresponding ligands and receptors in the liposome 1 and the liposome 2 are connected, and the result is obtained;

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

[0053] The present 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 nano drug delivery system.

[0054] After a large number of experiments and studies, the inventors found that the mass ratio of lecithin to ligands and receptors in the liposome has a very important influence on the successful connection of the two drug liposomes and the stability of the nano drug delivery system obtained after the connection, and screened and obtained the appropriate mass ratio of lecithin to ligands and receptors: 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); the mass ratio of lecithin to biotin in step S2 is (75-100): (1-5). The mass ratio of lecithin to ligands and receptors can enable liposome 1 encapsulating drug 1 and liposome 2 encapsulating drug 2 to be successfully and efficiently connected through ligands and receptors to obtain the nano drug delivery system, and ensure the stability of the nano drug delivery system so that it can play the expected role when entering the body.

[0055] Further, on the basis of successfully preparing the nano drug delivery system of the present invention and ensuring its stability, by further controlling the ratio of drug 1 and drug 2 in the above-mentioned nano drug delivery system, the chemotaxis and bactericidal performance of neutrophils to the infection site can be enhanced at the same time. The inventors have found through research that when the drug 1 is itaconic acid and the 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 nano drug delivery system of the present invention, so that it has a very good anti-infection effect.

[0056] The raw materials for preparing the liposome 1 encapsulating the drug 1 in the nano drug delivery system of the present invention include a fatty amine modified with poly(2-methacryloyloxyethyl phosphorylcholine) and a fatty amine modified with histidine, so that the surface of the prepared liposome 1 encapsulating the drug 1 can be modified with poly(2-methacryloyloxyethyl phosphorylcholine) (pMPC) and histidine (His). When the liposome 1 encapsulating the drug 1 enters the neutrophil, the liposome 1 encapsulating the drug 1 achieves lysosomal escape through the surface-modified histidine, enters the neutrophil cytoplasm in a complete form, is delivered to the infection site by the neutrophil and released through NETs; the modified positively charged pMPC can be affinity with the negatively charged LPS on the surface of the pathogen through electrostatic interaction, promotes the pathogen's uptake of it and releases the drug 1, and enhances the intracellular and extracellular antibacterial effects of NETs.

[0057] The conventional fatty amine for preparing liposomes in the art can be selected to be modified accordingly to obtain the fatty amine modified by the poly (2-methacryloxyethyl phosphorylcholine) and the fatty amine modified by histidine, or commercially available products can be purchased, and the nano drug delivery system of the present invention can be prepared. For example, in some embodiments, the fatty amine modified by the poly (2-methacryloxyethyl phosphorylcholine) is selected from the octadecylamine (pMPC-ODA) modified by the poly (2-methacryloxyethyl phosphorylcholine), the stearoylphosphatidylethanolamine (pMPC-DSPE) modified by the poly (2-methacryloxyethyl phosphorylcholine), the hydrogenated soybean phosphatidylcholine modified by the poly (2-methacryloxyethyl phosphorylcholine), and the dipalmitoylphosphatidylcholine modified by the poly (2-methacryloxyethyl phosphorylcholine). The fatty amine modified by the histidine is selected from the octadecylamine modified by the histidine, the stearoylphosphatidylethanolamine modified by the histidine, the hydrogenated soybean phosphatidylcholine modified by the histidine, and the dipalmitoylphosphatidylcholine modified by the histidine. Among them, poly(2-methacryloyloxyethylphosphorylcholine)-modified octadecylamine and histidine-modified octadecylamine are inexpensive and can effectively reduce the preparation cost, and thus can be preferably used.

[0058] The structural formula of the poly(2-methacryloyloxyethyl phosphorylcholine) modified octadecylamine is shown in the following formula I:

[0059]

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

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

[0062] In some embodiments, the structural formula 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 (when 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, the pMPC-ODA is prepared by the following method: dispersing ODA in a solvent, then dropping 2-bromoisobutyryl bromide into the mixture, stirring the reaction, washing, and removing the solvent to obtain an intermediate product; taking the intermediate product and MPC, adding them to a solvent, passing nitrogen to exclude oxygen, adding CuBr in a dark environment, stirring and reacting in a dark environment to obtain pMPC-ODA.

[0067] In some of the 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; 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 reacting Boc-His(Boc)-OH and ODA, and the reaction formula is as follows:

[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, and removing the organic solvent to obtain Boc-His(Boc)-ODA; adding the Boc-His(Boc)-ODA to dioxane hydrochloride, stirring the reaction, adjusting the pH value of the reaction mixture, and vacuum filtering to obtain His-ODA.

[0074] In some of the 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 7.5-8.5.

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

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

[0078] In some embodiments, the preparation method of the nano drug delivery system targeting neutrophils comprises the following steps:

[0079] S1, adding lecithin, cholesterol, poly (2-methacryloyloxyethyl phosphorylcholine) modified fatty amine, histidine modified fatty amine, ligand or receptor into organic solvent 1 to completely dissolve, removing organic solvent 1 to form a film; adding ultrapure water for hydration to obtain primary liposomes; ultrasonically crushing, filtering, to obtain liposomes 1; while stirring, dripping a solution containing drug 1 into the liposomes 1 to obtain liposomes 1 encapsulating drug 1;

[0080] S2, adding lecithin, cholesterol, dioleoylphosphatidylethanolamine, drug 2, and the receptor corresponding to the ligand added in step S1 or the ligand corresponding to the added receptor into organic solvent 2 to completely dissolve, removing organic solvent 2 to form a film; adding ultrapure water for hydration to obtain primary liposomes; ultrasonically crushing, filtering, to obtain liposomes 2; mixing the liposomes 2 with platelet membranes, ultrasonically dispersing, and then extruding the mixed solution through a membrane to obtain liposomes 2 encapsulating drug 2;

[0081] S3, mixing and incubating the liposome 1 encapsulating the drug 1 and the liposome 2 encapsulating the drug 2, and connecting the corresponding ligands and receptors in the liposome 1 and the liposome 2, thereby obtaining the product.

[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 crushing conditions in step S1 are: power 195W-325W, time 5min-7min, ultrasonication 1s-3s, and rest 2s-4s.

[0085] In some embodiments, the ultrasonic crushing conditions in step S2 are: power 195W-325W, time 5min-7min, ultrasonication 1s-3s, and rest 2s-4s.

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

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

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

[0089] In the following examples, IF-CLipos represents the nano drug 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 to 25KHz, the maximum ultrasonic power is 650W, and the power a%W represents a% of 650W, for example, 30%W is 650×30%W, that is, 195W.

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

[0092] (1) Synthesis and characterization of pMPC-ODA: 1.51 g ODA was dispersed in 100 mL chloroform and 1.4 mL triethylamine at room temperature for 30 min. Then 0.55 mL 2-bromoisobutyryl bromide was dropped into the mixture, and the mixture gradually became a transparent solution and was stirred at 40 °C for 12 h. The solution was washed with 100 mL 1 M hydrochloric acid solution and ultrapure water 3 times each, and the chloroform was completely removed by rotary evaporation to obtain a white intermediate product powder. 0.41 g intermediate product and 1.94 g MPC were put into a two-necked flask, connected to a condenser, and sealed with a rubber stopper, adhesive tape and sealing film. Vacuum and nitrogen were repeatedly introduced to remove oxygen. 8 mL dichloromethane, 8 mL ethanol and 600 μL pentamethyldiethylenetriamine were added with a syringe. After nitrogen was continued for 30 min, 0.21 g CuBr was added in the dark. The solution was stirred at 40 °C and kept in the dark overnight. The obtained solution was dialyzed with ethanol and water for 48 hours, and pMPC-ODA was obtained after freeze-drying. The obtained pMPC-ODA was dissolved in a 1:1 mixture of deuterated chloroform and deuterated methanol as a solvent, and TMS was used as an internal standard, and the structure was analyzed and characterized by nuclear magnetic resonance hydrogen spectrum.

[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 in 10 mL N,N'-dimethylformamide at room temperature, and stir for 2 h. After the reaction is completed, add 100 mL ultrapure water to mix well and continue stirring for 30 min. Collect the solid precipitate by vacuum filtration and dry in an oven at 40 °C overnight. The intermediate product is dissolved in 1 mL dichloromethane, then separated by column chromatography (petroleum ether: ethyl acetate = 2:1), and the remaining organic solvent is removed by rotary evaporation to obtain Boc-His(Boc)-ODA. All the Boc-His(Boc)-ODA is added to 3 mL of dioxane hydrochloride and stirred at room temperature for 30 min. After the reaction is completed, add 50 mL ultrapure water, and then adjust the pH value of the reaction mixture to 8 with 5% NaHCO3 solution while stirring. After stirring for 1 hour, vacuum filtration was performed, and the collected solid was placed in a 40°C oven and dried overnight to obtain His-ODA. The obtained His-ODA was dissolved in a 1:1 mixture of deuterated chloroform and tritiated methanol as a solvent, and TMS was used as an internal standard, and the structure was analyzed and characterized by nuclear magnetic resonance hydrogen spectrum.

[0094] (3) H NMR spectrum results ( Figure 1 ) shows that in pMPC-ODA 1 In the H-NMR spectrum, the b peak at the chemical shift δ=0.90ppm is the methyl proton signal (3H, CH3-CR-CH2-) of the hydrophobic tail of ODA, the c peak at δ=1.62ppm is the methylene proton signal (2H, -CH2-CR-CH3) at the connection between pMPC and ODA by atom transfer radical polymerization, and the d peak at δ=3.35ppm is the methyl proton signal (2H, -CH2-CR-CH3) of the tertiary amino 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 operation. 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 (molecular weight of about 1450) were filtered out, so the degree of polymerization of the final product was ≥4.

[0096] In the nuclear magnetic resonance hydrogen spectrum, the number of hydrogen atoms in each peak can be obtained by integrating and analyzing the characteristic peaks that appear. The characteristic peaks belonging to ODA and MPC are selected and compared with the number of hydrogen atoms originally contained to calculate how many ODA and pMPC are contained in a synthesized pMPC-ODA. After calculation, the 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 the chemical shift δ=0.90ppm is the methyl proton signal of the hydrophobic tail of ODA (3H, CH3-CR-CH2-), the f peak at δ=3.16ppm is the methylene proton signal next to the newly formed amide group on ODA (2H, -CH2-NH-CO-), and the h peak at δ=6.86ppm is the olefin hydrogen signal on the His imidazole ring (1H, CH=C), indicating the successful preparation of His-ODA.

[0098] Example 2 Prescription 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 at 60°C to fully dissolve, then vacuum evaporation to remove the organic solvent to form a thin film, add 6 mL of preheated ultrapure water and hydrate in a 60°C water bath for about 1 hour. Collect the obtained primary liposomes, optimize the particle size using a cell ultrasonic disruptor (ultrasonic conditions: power of 30% W, time of 5 min, interval of on 2 s and off 3 s), and pass the dispersion through 0.8 μm, 0.45 μm, and 0.22 μm filter membranes in turn for further homogenization to obtain pLipos.

[0100] (2) The results of the formulation screening of pMPC-ODA content and the ratio of chloroform to methanol mixed solvent are shown in Table 1. When pLipos was prepared with a mixed solvent of chloroform to methanol at a volume ratio of 1:1, the polydispersity index PDI of pLipos was greater than 0.3, the particle size distribution was large, and it was unstable. When the volume ratio was adjusted to 2:1, the PDI was less than 0.3, and the particle size distribution was good. Therefore, a mixed solvent of chloroform to methanol at a volume ratio of 2:1 was selected for the formulation of ITA-HpLipos. Since the later experiments required the positively charged pMPC-ODA to have affinity with the negatively charged LPS on the bacterial surface, thereby promoting the internalization of the pMPC-modified particles by 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 fully dissolve 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 hour. Collect the obtained primary liposomes and optimize the particle size using a cell ultrasonic disruptor (ultrasonic conditions: power of 30% W or 50% W, time of 5 min, interval of on 2 s and off 3 s), and pass the dispersion through 0.8 μm, 0.45 μm, and 0.22 μm filter membranes in turn for further homogenization to obtain HpLipos.

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

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

[0106]

[0107] (5) Preparation and characterization of ITA-HpLipos: Prepared by thin film dispersion method. Accurately weigh 150 mg of lecithin, 50 mg of cholesterol, 20 mg of pMPC-ODA, 10 mg of His-ODA and 500 μg of streptavidin, add 5 mL of methanol and 10 mL of chloroform to fully dissolve 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 hour. Collect the obtained primary liposomes and optimize the particle size using a cell ultrasonic disruptor (ultrasonic conditions: power of 50% W, time of 5 min, interval of on 2 s and off 3 s). The dispersion is further homogenized by passing through 0.8 μm, 0.45 μm, and 0.22 μm filter membranes in turn to obtain HpLipos (total volume 6 mL). pLipos does not contain His-ODA components, and the rest of the preparation method is the same as HpLipos. The Lipos component only contains 150 mg of lecithin and 50 mg of cholesterol, and the rest of the preparation method is the same as HpLipos. The loaded drug ITA is prepared by the pH gradient method. A 15 mg / mL ITA solution is prepared with a 1% glycine hydrochloride buffer solution with a pH value of 3.3, and then slowly dripped into HpLipos while stirring. The volume ratio of ITA solution to HpLipos is 1:2 (that is, 3 mL of ITA solution with a concentration of 15 mg / mL is added to 6 mL of HpLipos). After the addition is completed, it is placed in a 50°C water bath to react for 10 minutes, and then the reaction is terminated in an ice bath to obtain ITA-HpLipos. The particle size and potential are measured using a Malvern particle size analyzer to characterize the preparation.

[0108] (6) Particle size and potential results are as follows Figure 3 As shown, the particle size of ITA-HpLipos is 67.7±0.65nm; due to the absence of His-ODA, the Zeta potentials of Lipos and pLipos are -26.9±1.85mV and -20.1±1.45mV, respectively. After modification with His-ODA, the imidazole group can adsorb protons, so the Zeta potentials of HpLipos and ITA-HpLipos change from negative to positive, which are 8.9±1.80mV and 23.2±1.33mV, respectively.

[0109] Example 3 Prescription 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 fully dissolve in 60 μ, then vacuum evaporation to remove the organic solvent to form a thin film. Add 6 mL of preheated pH 7.4 PBS buffer and hydrate in a 60 μ water bath for about 1 hour. Collect the obtained primary liposomes and optimize the particle size using a cell ultrasonic disruptor (ultrasonic conditions: power of 30% W, time of 5 min or 7 min, interval of on 2 s and off 3 s), and pass the dispersion through 0.8 μm, 0.45 μm, and 0.22 μm filter membranes in turn for further homogenization to obtain DLipos.

[0111] (2) The results of the prescription screening of DOPE content and cell crusher ultrasonic time are shown in Table 3. When the cell crusher was ultrasonicated for 7 minutes, the polydispersity index PDI of DLipos was greater than 0.3, the particle size distribution was large, and it was unstable. When the ultrasonic time was adjusted to 5 minutes, the PDI was less than 0.3, and the particle size distribution was better. Therefore, the cell crusher ultrasonication for 5 minutes was selected for the prescription 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 drugs, a higher content of 10 mg DOPE was selected for the prescription of Fen-PDLipos.

[0112] Table 3 Effects of DOPE content and cell disruptor ultrasound 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 fully dissolve in 60 μm, then vacuum evaporation to remove the organic solvent to form a thin film. Add 6 mL of preheated hydration medium (PBS buffer at pH 7.4) and hydrate in a 60 μm water bath for about 1 hour. Collect the obtained primary liposomes. Use a cell ultrasonic disruptor to optimize the particle size (ultrasonic conditions: power of 30% W, time of 5 min, interval of on 2 s and off 3 s). The dispersion is passed through 0.8 μm, 0.45 μm, and 0.22 μm filter membranes in turn for further homogenization to obtain Lipo. In order to screen the hybridization conditions of 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 treated with water bath ultrasound (160W / 200W / 400W) for 20 min. The mixed solution after water bath ultrasound treatment or without treatment was repeatedly pushed through a 0.22 μm polycarbonate filter membrane 11 or 21 times through a liposome extruder to obtain platelet membrane hybrid liposomes.

[0116] (4) The prescription screening results of hybrid conditions are shown in Table 4 below. When the liposome and platelet membrane mixture is not pre-treated with water bath ultrasound, the polydispersity index PDI of the liposome is greater than 0.3, the particle size distribution is large, and it is unstable. After water bath ultrasound pre-treatment with 160W and 200W powers, the PDI of the prescription that passes through the membrane 11 times is greater than 0.3, and the PDI of the prescription that passes through the membrane 21 times is less than 0.3, and the stability is better. For the water bath ultrasound condition with a power of 400W, the PDI of passing through the membrane 11 times is better than that of passing through the membrane 21 times. After comprehensive comparison of several groups of prescriptions, the prescription with the smallest average PDI was selected, that is, the hybrid condition of pre-treating the liposome and platelet membrane mixture with 200W water bath ultrasound and passing it through a 0.22μm polycarbonate membrane 21 times was used for the prescription of Fen-PDLipos.

[0117] Table 4 Effects of water bath ultrasound power and extrusion membrane times 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 difference in the uptake of liposomes with different platelet membrane hybridization ratios by neutrophils, and then the appropriate hybridization ratio was screened. Accurately weigh 75 mg of lecithin and 25 mg of cholesterol, add 15 mL of chloroform and fully dissolve in 60 μ, then vacuum evaporation is performed to remove the organic solvent to form a thin film. Add 6 mL of preheated hydration medium (PBS buffer at pH 7.4) and hydrate in a 60 μ water bath for about 1 hour. Collect the obtained primary liposomes and use a cell ultrasonic disruptor to optimize the particle size (ultrasonic conditions: power of 30% W, time of 5 min, interval of on 2 s off 3 s). The dispersion was further homogenized by passing through 0.8 μm, 0.45 μm, and 0.22 μm filter membranes in turn to obtain Lipo. Platelet membrane hybrid liposomes PLipos were prepared according to the mass ratio of phospholipids to platelet membrane proteins of 1:500, 1:250, 1:100, and 1:50. After measuring the concentration of platelet membrane proteins, the platelet membrane solution was diluted to 100 μL according to the grouping, and the platelet membrane solution was treated with 200W water bath ultrasound for 20 minutes. 1 mL of Lipo was mixed with platelet membrane solutions of each concentration, and the mixed solution was repeatedly pushed through a 0.22 μm polycarbonate filter membrane 21 times through a liposome extruder to obtain PLipos. After preparing Lipo and PLipos with a mass ratio of phospholipids to platelet membrane proteins of 1:500, 1:250, 1:100, and 1:50, they were incubated with 10 mg / mL DiR dye in dark conditions for 40 minutes. Neutrophils were seeded in a 24-well plate at a density of 3×105 / well, and stained Lipo and PLipos were added in a dark plate (the final concentration of DiR in each well was 50 μg / mL, and the final concentration of phospholipids was 256 μg / mL). After incubation at 37°C for 1 h, the plates were washed twice with HBSS and resuspended in 500 μL 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 Figure 4 As shown in the figure, when the uptake time was 1h, the fluorescence intensity of PLipos with different hybridization ratios in neutrophils was significantly higher than that of the Lipo group, and as the hybridization ratio increased, the fluorescence intensity in neutrophils gradually increased, indicating that the presence of platelet membranes promoted the uptake of liposomes by neutrophils. The uptake of PLipos with hybridization ratios of 1:50 and 1:100 by neutrophils was significantly higher than that of other PLipos. Since there was no significant difference in the uptake of neutrophils by PLipos with a hybridization ratio of 1:50 and 1:100, in order to reduce the amount of platelet membrane used, a hybridization ratio of 1:100 was selected for the prescription of Fen-PDLipos.

[0121] (7) Preparation and characterization of Fen-PDLipos: Preparation by thin film dispersion method. Accurately weigh 75 mg of lecithin, 25 mg of cholesterol, 10 mg of DOPE, 24 mg of Fen and 1 mg of biotin, add 20 μL of oleic acid and 15 mL of chloroform to fully dissolve 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 at 60°C water bath for about 1 hour. Collect the obtained primary liposomes and optimize the particle size using a cell ultrasonic disruptor (ultrasonic conditions: power of 30% W, time of 5 min, interval of on 2 s and off 3 s). The dispersion is passed through 0.8 μm, 0.45 μm, and 0.22 μm filter membranes in turn for further homogenization to obtain Fen-loaded DOPE-modified liposomes (total volume of 6 mL). After measuring the platelet membrane protein concentration, the platelet membrane was diluted to 100 μL and ultrasonicated at 200 W in a water bath for 20 min. 6 mL of DOPE-modified liposomes loaded with Fen were mixed with 100 μL of platelet membrane solution, and the mixed solution was repeatedly pushed through a 0.22 μm polycarbonate filter 21 times by a liposome extruder to obtain Fen-PDLipos. PDLipos was not loaded with Fen, and the rest of the preparation method was the same as Fen-PDLipos. The particle size and potential were measured using a Malvern particle size analyzer to characterize the preparation.

[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 close, ranging from -10mV to -20mV.

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

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

[0125] (2) Morphological characterization of IF-CLipos: First, the morphology of IF-CLipos was investigated by transmission electron microscopy. After the preparation of IF-CLipos, 10 μL was dropped on the copper mesh and allowed to stand for 1 min to dry. Then, 3% phosphotungstic acid negative staining solution was dropped on the copper mesh and negatively stained for 20 s. The liquid was absorbed by filter paper and its morphology was observed under a transmission electron microscope at an operating voltage of 120 kV. In order to investigate the morphology of IF-CLipos by laser confocal microscopy, DiD-HpLipos and DiI-PDLipos were prepared. The concentrations of DiD and DiI were both 16.7 μg / mL. The two liposomes were mixed in a volume ratio of 1:1 and incubated at 37°C for 30 min to connect. A clean glass slide was taken and 5 μL of anti-fluorescence quenching sealing 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 100 times.

[0126] (3) The morphological characterization results of IF-CLipos are as follows Figure 6 As shown, ITA-HpLipos and Fen-PDLipos are connected to form IF-CLipos through the non-covalent interaction of avidin and biotin, and both have a typical liposome phospholipid bilayer structure, regular shape, and are connected in a "hand-in-hand" manner. Figure 7 Representative fluorescence images of IF-CLipos under confocal laser scanning microscope.

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

[0128] (1) The uptake of PDLipos, HpLipos and CLipos by neutrophils in the systemic circulation of mice was investigated by flow cytometry to evaluate the hijacking ability of IF-CLipos on neutrophils. According to the above steps, Cou6-HpLipos and DiR-PDLipos were prepared and connected to obtain CLIpos, in which the concentrations of Cou6 and DiR were both 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 with 100 μL of 50 μg / mL PE-Ly6G through the tail vein to mark neutrophils in the systemic circulation. Subsequently, 100 μL of the corresponding liposomes were injected into the tail vein of each group of mice except the blank group. After the mice were protected from light for 1 hour, fresh blood was taken 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 8) showed that PDLipos, due to the platelet membrane hybridization, can be recognized and phagocytosed by neutrophils in the systemic circulation, with an uptake rate of 16.61±0.60%; while for HpLipos without platelet membrane hybridization, the neutrophil uptake rate was only 0.25±0.13%; when PDLipos and HpLipos are connected to form CLIpos, HpLipos can hijack neutrophils through PDLipos, and the neutrophil uptake rate of CLIpos is 7.63±0.60%. The above results show that IF-CLipos can hijack neutrophils, among which Fen-PDLipos recognizes and binds to neutrophils through platelet membranes, and ITA-HpLipos is taken up by neutrophils together with Fen-PDLipos through the interaction between biotin and avidin.

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

[0131] (1) ITA-HpLipos and Fen-PDLipos were prepared according to the method described in Examples 2 and 3. 100 μL of each liposome was added to 900 μL of PBS buffer with pH values ​​of 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 potentiometric cup. The particle size and potential were measured using a Malvern nanoparticle sizer.

[0132] (2) Experimental results Fig. 9 ) showed that as the acidity increased, the particle size of ITA-HpLipos remained almost unchanged, and the Zeta potential changed from negative to positive and gradually increased. This is because His-ODA has a strong nucleophilic group imidazole ring, which can be continuously protonated in an acidic environment, making the surface of ITA-HpLipos positively charged; while when the pH value of Fen-PDLipos dropped to 5.5 and 4.5, that is, in a simulated lysosomal environment, the particle size of Fen-PDLipos suddenly increased, the liposome state was no longer stable, and the absolute value of the Zeta potential decreased. This is because the hydroxyl end of DOPE on Fen-PDLipos was protonated in an acidic environment, resulting in the neutralization of the negative charge originally carried by the liposome, and DOPE formed a hexagonal crystal phase, thereby causing the fusion of the liposome membrane. This result shows that ITA-HpLipos can adsorb H through His-ODA + The pH in the lysosome increases, and then H + With Cl - A large amount of water is pumped into the lysosome, causing the lysosome to rupture and achieve lysosomal escape. At the same time, ITA-HpLipos itself can remain stable and not be destroyed, while 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 Education Function of IF-CLipos

[0134] (1) Immunofluorescence staining was used to investigate the effect of Fen-PDLipos in IF-CLipos on the expression of CXCR2 receptor protein in neutrophils. Neutrophils were extracted from mouse bone marrow and cultured at 8×10 5 / well were inoculated into a 24-well plate containing poly-lysine slides. LPS was added to each group except the blank group at a final concentration of 10 μg / mL. Free fenofibrate solution was prepared with DMSO. Fen-PDLipos was prepared as described above. Fenofibrate and Fen-PDLipos were administered at a final concentration of 40 μg / mL per well. 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 minutes, and then place at room temperature for 20 minutes. Weigh 0.5g BSA and dissolve it in 50mL PBS buffer, then add 50μL Tween 20 and vortex to dissolve, and obtain 1% BSA blocking solution. After fixation, drain the liquid and wash three times with PBS, add 500μL blocking solution, and place at 37℃ for 30 minutes.

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

[0137] (4) Experimental results Fig.10 ) showed that after 1 h of stimulation with 10 μg / mL LPS, 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, which could significantly inhibit the internalization of CXCR2 receptor protein on the surface of neutrophils and restore its expression level to a level close to that of normal neutrophils.

[0138] Example 8 Neutrophils release ITA-HpLipos through NETs to clear extracellular bacteria

[0139] (1) The antibacterial effect of IF-CLipos on NETs was investigated by measuring the growth curves of E. 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. Neutrophils were extracted from mouse bone marrow and the cell density was adjusted to 2×10 5 / mL, add PMA with 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 an ITA concentration of 2560μg / mL and incubate for 2h, then add PMA with a final concentration of 100nM and incubate at 37℃ in the dark for 4h.

[0140] (2) E. coli and P. aeruginosa bacterial suspensions were diluted to 1×10 5 CFU / mL, 100 μL per well was plated on a 96-well plate. 100 μL LB liquid culture medium was added to each well of the control group, 50 μL neutrophil NETs suspension and 50 μL LB liquid culture medium were added to each well of the NETs group, 50 μL IF-CLipos and 50 μL LB liquid culture medium were added to each well of the IF-CLipos group, and 100 μL neutrophil NETs suspension containing IF-CLipos was added to each well of the NETs+IF-CLipos group (the final concentration of itaconic acid was 640 μg / mL, and the ratio of neutrophils to bacterial single colonies was 1:1).

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

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

[0143] (4) Experimental results Fig.11) showed that the growth multiple of E. coli in the Control group was 31.2±3.2 times at 48h, while that of P. aeruginosa was 40.8±2.0 times; IF-CLipos can enhance the inhibition of NETs on bacterial growth. After administration of NETs+IF-CLipos group, the growth multiple of E. coli at 48h was 4.7±1.8 times, and the growth multiple of P. aeruginosa at 48h was 10.1±1.0 times, which was significantly lower than the bacterial growth rate of Control, and better than the effect of NETs and IF-CLipos alone. It shows that compared with NETs released by neutrophils, the extracellular antibacterial effect of NETs released after incubation of IF-CLipos with neutrophils is significantly enhanced, and is better than the extracellular antibacterial effect of IF-CLipos itself.

[0144] Example 9 Neutrophils release ITA-HpLipos through NETs to clear intracellular bacteria

[0145] (1) IF-CLipos was prepared according to the method described in Example 4 and diluted to an itaconic acid concentration of 1280 μg / mL for later use. Neutrophils were extracted from mouse bone marrow and the cell density was adjusted to 1×10 7 / mL, add PMA with a final concentration of 100 nM and incubate at 37°C in the dark for 4 h. Take another 500 μL neutrophil suspension, add 500 μL IF-Clipos with an ITA concentration of 1280 μg / mL and incubate for 2 h, then add PMA with a final concentration of 100 nM and incubate at 37°C in the dark for 4 h to induce the formation of NETs.

[0146] (2) RAW264.7 macrophages were cultured at a density of 5×10 5 / well inoculated into a 24-well plate containing a normal cell slide and cultured overnight at 37°C and 5% CO2. After obtaining the S. typhimurium bacterial solution, dilute the bacterial solution to 1×10 7 CFU / mL, 500μL of bacterial solution was added to a 24-well plate and incubated with RAW264.7 macrophages for 1h. At this time, the ratio of bacterial colonies to macrophages was 10:1. After incubation, the supernatant was discarded and DMEM medium containing 100μg / mL gentamicin was added and incubated for 1h to remove extracellular bacteria. The supernatant was discarded and washed three times with PBS to obtain the model of Salmonella typhimurium invading macrophage intracellular bacteria.

[0147] (3) The culture supernatant of RAW264.7 macrophages was discarded and the drugs were administered according to the grouping. 1 mL of complete DMEM medium was added to each well of the control group, 500 μL of neutrophil NETs suspension and 500 μL of complete DMEM medium were added to each well of the NETs group, 500 μL of IF-CLipos and 500 μL of complete DMEM medium (the final concentration of itaconic acid was 640 μg / mL) were added to each well of the IF-CLipos group, and 1 mL of neutrophil NETs suspension containing IF-CLipos was added to each well of the NETs+IF-CLipos group. At this time, the ratio of neutrophil number to bacterial single colony number to macrophage number was 10:10:1. After drug administration, the cells were incubated at 37°C and 5% CO2 for 12 h.

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

[0149] (5) Experimental results Fig.12 ) showed that the NETs+IF-CLipos group had an antibacterial effect superior to that of NETs alone, and the macrophage lysate containing intracellular bacteria had almost no colonies growing on the agar plate, and the number of intracellular residual S. typhimurium was less than 1 after quantifying the number of colonies in a single macrophage. This indicates that compared with the NETs released by neutrophils, the intracellular antibacterial effect of the NETs released after the IF-CLipos of the present invention was incubated with neutrophils was significantly enhanced, and was superior to the intracellular antibacterial effect of IF-CLipos itself.

[0150] Example 10

[0151] The mouse acute intestinal infection model was established by oral 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 deprivation of water for 6 hours, the mice were first gavaged with 100 μL of 5% NaHCO3 solution to neutralize gastric acid. 30 minutes later, the healthy group mice were gavaged with 400 μL of normal saline, and the model group and the treatment group mice were gavaged with 400 μL of 1×10 9CFU / mL of Salmonella typhimurium liquid. Fen-PDLipos and IF-CLipos were prepared according to the methods of Examples 3 and 4, and Fen-PDLipos and IF-CLipos were injected into the tail vein according to the grouping and Fen dosage of 25 mg / kg. 48 hours after modeling and administration, the mice were killed by cervical dislocation and dissected, and the jejunum segments of the mouse small intestine of appropriate length were selected, fixed with 4% paraformaldehyde, embedded in paraffin after gradient dehydration, and stained with Ly6G fluorescent antibody after sectioning, and observed under 10 times of integrated cell imager.

[0152] Experimental results ( Fig.13 ) showed that compared with the Model group, the green fluorescence signal of neutrophils in the infected part of the mouse intestine was significantly increased in the Fen-PDLipos and IF-CLipos groups after treatment, which means that the chemotaxis of neutrophils to the infected lesions can be restored through "training", thereby increasing neutrophil infiltration and promoting the clearance of pathogens. Moreover, the effect of IF-CLipos in restoring the chemotaxis of neutrophils to the infected lesions is better than that of the Fen-PDLipos group, and the green fluorescence signal is stronger.

[0153] Embodiment 11

[0154] The acute intestinal infection model of mice was established and grouped according to the method of Example 10, and the drugs were injected into the tail vein according to the grouping. The mice were killed by cervical dislocation and dissected, and the small intestine jejunum segment of appropriate length was selected, fixed with 4% paraformaldehyde, gradient dehydrated, paraffin embedded, and sliced ​​for rapid Gram staining, and the amount of bacteria was observed under 10 times of the integrated cell imager.

[0155] Experimental results ( Fig.14 ) showed that in the Model group, due to the presence of a large number of bacteria in the intestinal infection, although Fen-PDLipos treatment could increase the infiltration of neutrophils in the infected area and thus have antibacterial effects to a certain extent, some bacteria still remained; and after IF-CLipos treatment, there were almost no pathogens remaining in the intestines of mice, indicating that the therapeutic effect of IF-CLipos was significantly better than that of Fen-PDLipos. This may be because after IF-CLipos trained neutrophils to chemotaxis to the infected area of ​​the intestine, neutrophils were stimulated by pathogens to form NETs containing ITA-HpLipos, which were taken up by pathogens and released itaconic acid, thereby enhancing the effect of NETs in clearing pathogens.

[0156] Example 12

[0157] The mouse acute intestinal infection model was established by the method of Example 10, and 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 of Examples 2 and 3, but streptavidin and biotin were not added during the preparation process; IF-CLipos was prepared according to the method of Example 4. The drugs were injected into the tail vein according to the grouping. The Fen-PDLipos combined with ITA-HpLipos treatment group was injected with Fen at a dose of 25 mg / kg into the tail vein, and ITA-HpLipos was injected with an ITA dose of 30 mg / kg into the tail vein: the IF-CLipos treatment group was injected with Fen at a dose of 25 mg / kg, and the ITA dose was 30 mg / kg. The mice were killed by cervical dislocation and dissected. The small intestine jejunum segment of appropriate length was selected and fixed with 4% paraformaldehyde. After gradient dehydration, paraffin embedding and sectioning, rapid Gram staining was performed and the amount of bacteria was observed under 10 times magnification of the integrated cell imager.

[0158] The experimental results showed that there were a large number of bacteria in the intestine of the Model group. Both the Fen-PDLipos combined with ITA-HpLipos treatment group and the IF-CLipos treatment group had significant therapeutic effects. However, there were a small amount of bacteria remaining in the intestine of the mice in the Fen-PDLipos combined with ITA-HpLipos treatment group, while there were almost no bacteria remaining in the intestine of the mice in the IF-CLipos treatment group, indicating that the therapeutic effect of the IF-CLipos treatment group was better than that of the Fen-PDLipos combined with ITA-HpLipos treatment group. This may be because ITA-HpLipos in the IF-CLipos treatment group can be taken up by neutrophils together with Fen-PDLipos through the connection between streptavidin and biotin, and then driven to the site of infection with neutrophils, and exert its effect through the NETs released by neutrophils, thereby enhancing the anti-infection effect; while in the Fen-PDLipos combined with ITA-HpLipos treatment group, ITA-HpLipos is difficult to be taken up by neutrophils, cannot be driven to the site of infection, and exert its effect through the released NETs.

[0159] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for preparing a nano drug delivery system targeting neutrophils, characterized in that: The following steps are involved: S1, adding lecithin, cholesterol, poly (2-methacryloyloxyethyl phosphorylcholine) modified fatty amine, histidine modified fatty amine, ligand or receptor into organic solvent 1 to prepare liposome 1; dropping a solution containing drug 1 into the liposome 1 while stirring to obtain liposome 1 encapsulating drug 1; the drug 1 is an anti-inflammatory drug; S2, adding lecithin, cholesterol, dioleoylphosphatidylethanolamine, drug 2, and a receptor or ligand corresponding to the ligand or receptor added in step S1 into an organic solvent 2 to prepare liposome 2; mixing the liposome 2 with a platelet membrane to prepare a liposome 2 encapsulating drug 2; the drug 2 is a drug that induces neutrophil chemotaxis to the infection site; S3, mixing and incubating the liposome 1 encapsulating the drug 1 and the liposome 2 encapsulating the drug 2, so that the corresponding ligands and receptors in the liposome 1 and the liposome 2 are connected, and the result is obtained; The ligand and receptor are biotin and streptavidin, respectively.

2. The preparation method according to claim 1, characterized in that In step S1, streptavidin is added, and in step S2, biotin is added; Preferably, in step S1, the mass ratio of lecithin to streptavidin is (150-200): (0.5-3); and / or, Preferably, in step S2, the mass ratio of lecithin to biotin is (75-100):(1-5).

3. The preparation method according to any one of claims 1 to 2, characterized in that: 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.

4. The preparation method according to claim 3, characterized in that: The drug 1 is itaconic acid, and the drug 2 is fenofibrate; Preferably, in step S1, the mass ratio of lecithin to itaconic acid is (150-200): (9-45); and / or, Preferably, in step S2, the mass ratio of lecithin to fenofibrate is (75-100): (20-30).

5. The preparation method according to claim 1 or 4, characterized in that: In step S3, the liposome 1 encapsulating the drug 1 and the liposome 2 encapsulating the drug 2 are mixed and incubated at a mass ratio of the drug 1 to the drug 2 of (6-30):(20-30).

6. The preparation method according to claim 1, characterized in that: The poly(2-methacryloxyethylphosphorylcholine)-modified fatty amine is selected from poly(2-methacryloxyethylphosphorylcholine)-modified octadecylamine, poly(2-methacryloxyethylphosphorylcholine)-modified stearoylphosphatidylethanolamine, poly(2-methacryloxyethylphosphorylcholine)-modified hydrogenated soybean phosphatidylcholine, poly(2-methacryloxyethylphosphorylcholine)-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.

7. The preparation method according to claim 6, characterized in that: 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; 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), preferably 1:(50-250), and more preferably 1:(50-100).

8. A nano drug delivery system targeting neutrophils, characterized in that: The method is prepared according to any one of claims 1 to 7.

9. Use of the neutrophil-targeted nano drug delivery system as claimed in claim 8 in the preparation of anti-infective drugs.

10. An anti-infective drug, characterized in that: It comprises an active ingredient and a pharmaceutically acceptable carrier; the active ingredient comprises the nano drug delivery system targeting neutrophils as claimed in claim 8.

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