A nano antibacterial drug

Abstract

The present invention relates to an antibacterial nano drug, belonging to the field of medical technology. The antibacterial nano drug comprises: a nano skeleton, and a leukocyte cell membrane located at the outermost layer of the antibacterial nano drug. The antibacterial nano drug has the ability of leukocytes to capture and phagocytize bacteria, but compared with leukocytes, since it does not contain a cell nucleus, it will not be destroyed and degraded by DNA enzymes, nor will it cause inflammatory factor storms and tissue damage. In particular, when the outermost layer of the antibacterial nano drug is a neutrophil cell membrane and loaded with NETs inhibitory drugs, it can be targeted to neutrophils, and the NETs inhibitory drugs are used to act on neutrophils, appropriately inhibiting the production of endogenous NETs, ​​and avoiding damage to the body caused by excessive NETs. The antibacterial nano drug has broad application prospects in the field of antibacterial, especially in the field of sepsis treatment.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a nano antibacterial drug. Background Art

[0002] Sepsis refers to the dysregulation of the body's immune response to infection, which leads to life-threatening organ dysfunction and even death. The root cause of sepsis is severe infection, which is usually caused by bacteria. In recent years, basic theories and clinical treatment methods in the field of sepsis have made certain progress, but the morbidity and mortality of sepsis patients have not been fundamentally improved. According to relevant statistics, there are approximately 48.9 million new cases of sepsis patients worldwide each year, and approximately 11 million deaths from sepsis each year, accounting for 19.7% of the total global deaths each year. Therefore, in-depth research on the pathogenesis of sepsis and finding more effective prevention and treatment methods have become scientific issues that need to be urgently addressed in the field of sepsis.

[0003] Neutrophils originate from hematopoietic stem cells in the bone marrow and differentiate from common myeloid progenitor cells, participating in the formation of the body's most important natural immune defense line. Normally, after neutrophils differentiate and mature, they migrate from the bone marrow to the blood and reside in the blood to participate in circulation. When sepsis occurs, the number of neutrophils released from the bone marrow into the blood increases rapidly in a short period of time; neutrophils are activated in large numbers and quickly migrate to the infected or lesion area, inhibiting pathogens through a variety of action modes, including chemotaxis, phagocytosis, granulase release, pro-inflammatory factor release, etc.; in addition, they can also capture, fix and kill pathogens by forming neutrophil extracellular traps (NETs). However, if the infection cannot be controlled in time, neutrophils will produce a large number of inflammatory factors, a large number of NETs, ​​etc., which will seriously damage the surrounding tissue cells and aggravate the body's immune dysfunction, which may aggravate the condition of sepsis.

[0004] In recent years, nanomedicine has attracted more and more attention due to its unique advantages in improving treatment efficiency. Nanomaterials not only have adjustable physical and chemical properties (such as particle size, surface charge and chemical composition), but also have multiple biological functions (such as anti-inflammatory, antibacterial and targeted drug delivery, etc.), and are widely used in the biomedical field, including the diagnosis and treatment of sepsis. Yan et al. reported a hollow mesoporous polydopamine nanoparticle loaded with nicotinamide adenine dinucleotide (NAD+) and calcium ion chelator (BAPTA-AM), which can scavenge reactive oxygen species, chelate Ca 2+, and restore cellular energy homeostasis by supplementing NAD+, thereby achieving a therapeutic effect on animal models of sepsis. However, existing nanomedicines for the treatment of sepsis mainly focus on clearing extracellular inflammatory factors, targeted delivery of antibacterial drugs, or regulating the polarization of macrophages, but are unable to clear NETs formed by neutrophils. After NETs are destroyed and degraded by DNA enzymes in the blood circulation, NETs degradation products including histones, free nucleic acids, and various proteases are generated. These degradation products are highly destructive. On the one hand, they can stimulate a variety of immune cells to release a large number of inflammatory factors, aggravate the inflammatory response in the sepsis body, and further disrupt the immune system; on the other hand, the proteases therein will directly damage the surrounding tissue cells. Summary of the invention

[0005] In order to solve the problem that existing drugs cannot solve the problem of excessive production of NETs under conditions of severe bacterial infection, the present invention provides an antibacterial nanodrug, which is a leukocyte biomimetic drug. The outermost layer is the leukocyte cell membrane, which has the ability of leukocytes to capture and phagocytize bacteria, but does not contain a cell nucleus, will not be destroyed and degraded by DNA enzymes, and will not cause inflammatory factor storms and tissue damage.

[0006] Furthermore, the antibacterial nanomedicine uses multi-layer nanosheets as nano-skeleton, loads NETs inhibitory drugs, and the outermost layer is the neutrophil cell membrane. The antibacterial nanomedicine can target and inhibit the excessive production of NETs by neutrophils, avoiding excessive NETs from exacerbating inflammatory reactions and damaging tissues. After the nano-skeleton of the antibacterial nanomedicine is unfolded, it forms a mesh structure similar to the DNA skeleton in NETs, ​​simulating the structure and function of NETs and improving the antibacterial effect.

[0007] The technical solution provided by the present invention is specifically as follows:

[0008] In a first aspect, the present invention provides an antibacterial nanomedicine, which comprises: a nanoskeleton, and a leukocyte cell membrane located at the outermost layer of the antibacterial nanomedicine.

[0009] The outermost leukocyte cell membrane of the antibacterial nano drug provided by the present invention is one or more of the macrophage cell membrane, the neutrophil cell membrane, the basophil cell membrane, and the eosinophil cell membrane, and has the ability of leukocytes to capture and engulf bacteria, but does not contain a nucleus, will not be destroyed and degraded by DNA enzymes, and will not cause inflammatory factor storms and tissue damage. And the leukocyte cell membrane can enhance the stability of the antibacterial nano drug in liquid. The antibacterial nano drug can be used in combination with other antibacterial active factors, using the antibacterial nano drug to fix bacteria, and using other antibacterial active factors to eliminate bacteria.

[0010] Furthermore, the antibacterial nano-drug comprises: a nano-skeleton, an antibacterial drug loaded on the nano-skeleton, and a leukocyte cell membrane located at the outermost layer of the antibacterial nano-drug.

[0011] After the antibacterial nanomedicine captures and engulfs bacteria in the leukocyte membrane, it uses the antibacterial drug loaded in the inner layer to kill bacteria, thereby enhancing the antibacterial effect. The nanoskeleton has a large specific surface area and a small volume, and can load a large amount of antibacterial drugs and leukocyte membranes, and can freely pass through body fluids. The leukocyte membrane is set on the outermost layer of the antibacterial nanomedicine to achieve the sustained release of the antibacterial drug and prolong its half-life in the body.

[0012] In some embodiments of the present invention, the antibacterial nanomedicine includes: a nanoskeleton, a NETs inhibitory drug and / or an antibacterial drug loaded on the nanoskeleton, and a neutrophil cell membrane located at the outermost layer of the antibacterial nanomedicine.

[0013] The outermost neutrophil cell membrane of the antibacterial nanodrug retains the function of neutrophils, can capture bacteria, has the ability to target neutrophils, and can enhance the stability of the antibacterial nanodrug in liquids; after the antibacterial nanodrug reaches the neutrophils, the NETs inhibitory drug acts on the neutrophils to appropriately inhibit the excessive production of NETs; after the antibacterial nanodrug captures bacteria, the antibacterial drug kills the captured bacteria.

[0014] In some embodiments of the present invention, the size of the antibacterial nanomedicine is 100-500 nm, preferably 200-400 nm, and more preferably 250-300 nm. In a specific embodiment, the size of the antibacterial nanomedicine is 261.633±4.964 nm.

[0015] In some embodiments of the present invention, the nano skeleton comprises one or more of nanoparticles, nanowires, monolayer nanosheets, multilayer nanosheets, and nanofilms. These nanomaterials have a large specific surface area and a small volume, and can load a large amount of antibacterial drugs, NETs inhibitory drugs, and neutrophil cell membranes, and can freely travel in body fluids after loading antibacterial drugs, NETs inhibitory drugs, and neutrophil cell membranes. Preferably, the nano skeleton uses nanomaterials such as nanoparticles, monolayer nanosheets, and multilayer nanosheets, which are formed by self-assembling antibacterial drug layers, NETs inhibitory drug layers, and neutrophil cell membrane layers, and the nano drug is between a cube and a sphere.

[0016] In some embodiments of the present invention, the nano-skeleton comprises a multi-layer nano-sheet or only a multi-layer nano-sheet. Preferably, the nano-skeleton is a multi-layer nano-sheet. The multi-layer nano-sheet contains a multi-layer folded structure. When the multi-layer nano-sheet is self-assembled layer by layer, the antibacterial drug, the NETs inhibitory drug, and the neutrophil cell membrane enter between the sheets. Compared with nanoparticles and nanowires, the multi-layer nano-sheet has a larger load, and the multi-layer nano-sheet can be unfolded in body fluids to form a mesh structure similar to the DNA skeleton in NETs, ​​which not only increases the exposure area of ​​the antibacterial drug, the NETs inhibitory drug, and the neutrophil cell membrane attached to its surface, but also can capture bacteria, and synergistically improve the bactericidal ability of antibacterial nano-drugs.

[0017] In some embodiments of the present invention, the multilayer nanosheets include titanium carbide nanosheets or only titanium carbide nanosheets. Preferably, the multilayer nanosheets are titanium carbide nanosheets. Titanium carbide nanosheets have rich surface groups and are easy to modify. Titanium carbide has a certain antibacterial ability. Therefore, the mesh structure of the titanium carbide nanosheets after unfolding not only has the ability to capture bacteria, but also has the ability to kill bacteria.

[0018] In some embodiments of the present invention, the antibacterial drug comprises chitosan quaternary ammonium salt or only uses chitosan quaternary ammonium salt, preferably, the antibacterial drug is chitosan quaternary ammonium salt. The surface of chitosan quaternary ammonium salt is rich in hydroxyl and quaternary ammonium groups, which can adsorb and fix bacteria and has a strong bactericidal effect.

[0019] In some embodiments of the present invention, the NETs inhibitory drug comprises 4-octyl itaconic acid or is only 4-octyl itaconic acid. Preferably, the NETs inhibitory drug is 4-octyl itaconic acid. 4-octyl itaconic acid can act on neutrophils to inhibit the excessive production of NETs, ​​and has a bactericidal effect, and can cooperate with antibacterial drugs to kill bacteria.

[0020] In some embodiments of the present invention, the mass ratio of the nanoskeleton, the antibacterial drug, the NETs inhibitory drug, and the neutrophil cell membrane is 1.0:2.0-2.5:3.0-3.5:6.0-6.5.

[0021] In a second aspect, the present invention provides an antibacterial composition comprising the above antibacterial nanomedicine as an active ingredient, and optionally one or more pharmaceutically acceptable excipients.

[0022] In some embodiments of the present invention, the antibacterial nano drug is used as an antibacterial active ingredient, but not the only active ingredient. Preferably, the antibacterial nano drug is used as the only antibacterial active ingredient in the antibacterial composition, and the antibacterial composition does not contain other antibacterial active ingredients.

[0023] In some embodiments of the present invention, the antibacterial composition is an injection preparation, a respiratory inhalation, a respiratory instillation, and preferably an intravenous injection preparation.

[0024] In a third aspect, the present invention provides a method for preparing an antibacterial nano drug, wherein a leukocyte cell membrane layer is self-assembled on the outermost layer of the antibacterial nano drug to obtain the antibacterial nano drug.

[0025] In some embodiments of the present invention, an antibacterial drug layer and / or a NETs inhibitory drug layer and a neutrophil cell membrane layer are self-assembled layer by layer on the surface of the nanoskeleton to obtain the antibacterial nanomedicine.

[0026] In some embodiments of the present invention, an antimicrobial drug layer and / or a NETs inhibitory drug layer and a neutrophil cell membrane layer are self-assembled layer by layer on the surface of the nanoskeleton, comprising: sequentially contacting the nanoskeleton with the antimicrobial drug and / or NETs inhibitory drug and the neutrophil cell membrane in a solution to perform layer-by-layer self-assembly.

[0027] In some embodiments of the present invention, the step of sequentially contacting the nano-skeleton with the antimicrobial drug and / or NETs inhibitory drug and the neutrophil cell membrane in a solution to perform layer-by-layer self-assembly comprises:

[0028] The antibacterial drug is brought into contact with the nano-skeleton in a solvent and self-assembled to obtain the nano-skeleton modified with the antibacterial drug; the mass ratio of the antibacterial drug to the nano-skeleton is 2.0-2.5:1.0;

[0029] The NETs inhibitory drug and the nano-skeleton modified with the antibacterial drug are brought into contact with each other in a solvent and self-assembled to obtain composite nanoparticles loaded with the NETs inhibitory drug layer; the mass ratio of the NETs inhibitory drug to the nano-skeleton modified with the antibacterial drug is 1.0-1.5:1.0;

[0030] The neutrophil cell membrane and the composite nanoparticles loaded with the NETs inhibitory drug layer are brought into contact in a solvent and self-assembled to obtain a nanomedicine for treating sepsis; the mass ratio of the neutrophil cell membrane and the composite nanoparticles loaded with the NETs inhibitory drug layer is 1.0-1.5:1.0.

[0031] In some embodiments of the present invention, the neutrophil cell membrane, antimicrobial drugs, nano-skeleton, and NETs inhibitory drugs have opposite charges and are self-assembled layer by layer through electrostatic adsorption.

[0032] In some embodiments of the present invention, the nano-skeleton is a titanium carbide nano-sheet, which is negatively charged and can adsorb positively charged antibacterial drugs for self-assembly; and / or

[0033] The antibacterial drug is quaternary ammonium salt chitosan, which is positively charged and can be self-assembled on the negatively charged nano-skeleton, and the negatively charged NETs inhibitory drug is assembled on the quaternary ammonium salt chitosan layer; and / or

[0034] The NETs inhibitory drug is 4-octylitaconic acid, which is negatively charged and can be self-assembled on the positively charged antibacterial drug layer.

[0035] In some embodiments of the present invention, the method for preparing the antibacterial nanomedicine provided by the present invention further comprises: providing a neutrophil cell membrane, wherein providing the neutrophil cell membrane comprises:

[0036] Isolating cell membranes of neutrophils from the blood of patients with sepsis; and / or

[0037] Isolate the cell membranes of neutrophils from mouse blood.

[0038] In some embodiments of the present invention, the method for preparing the antibacterial nanomedicine provided by the present invention further comprises providing a nano-skeleton, wherein the nano-skeleton is a titanium carbide nano-sheet; the step of providing the nano-skeleton comprises:

[0039] Ti3AlC2 powder was slowly added to LiF / HCl etching solution under ice bath conditions;

[0040] Raise the temperature to allow the etching reaction to proceed slowly, and stop the reaction after a stable suspension is obtained;

[0041] The solid product is washed and dried to obtain titanium carbide nanosheets.

[0042] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0043] 1. Compared with existing antibacterial nanomedicines, the antibacterial nanomedicine provided by the present invention has the ability of leukocytes to capture and remove bacteria because its outermost layer is the leukocyte cell membrane. However, compared with leukocytes, it does not contain a cell nucleus and will not be destroyed and degraded by DNA enzymes, nor will it release free nucleic acids (cfDNA), elastase (NE), myeloperoxidase (MPO) and histones, etc., which may cause tissue damage in the body. Therefore, it will not trigger inflammatory factor storms and tissue damage.

[0044] 2. The antibacterial nanomedicine provided by the present invention has the ability to target neutrophils. After reaching the neutrophils, it can exert the function of NETs inhibitory drugs to inhibit excessive production of NETs, ​​thereby effectively inhibiting the excessive production of NETs and avoiding tissue damage caused by excessive production of NETs. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0046] Figure 1 The scanning electron microscope images of MXene nanosheets and M / C / OI / C nanoparticles in Test Example 1;

[0047] Figure 2 is the average particle size of MXene, M / C, M / C / OI, and M / C / OI / C nanoparticles in Test Example 2;

[0048] Figure 3 The change curve of particle size and dispersion index (PDI) of M / C / OI / C nanoparticles in test example 2 in ultrapure water over a period of 7 days;

[0049] Figure 4 is the average zeta potential of MXene, M / C, M / C / OI, and M / C / OI / C nanoparticles in Test Example 2;

[0050] Figure 5 The images of neutrophil cell membrane, M / C / OI and M / C / OI / C nanoparticles in Test Example 3 were subjected to SDS polyacrylamide gel electrophoresis followed by Coomassie brilliant blue staining;

[0051] Figure 6 The hemolysis rates of 4-OI, M / C / C NPs, and M / C / OI / C NPs in Test Example 4 (the concentration of each group was 20 μg / mL);

[0052] Figure 7 This is the toxicity experiment of different concentrations of 4-OI, M / C / C NPs, and M / C / OI / C NPs on mouse alveolar epithelial cells MLE12 in Test Example 5;

[0053] Figure 8 This is the toxicity experiment of 4-OI, M / C / C NPs, and M / C / OI / C NPs with different incubation times on mouse alveolar epithelial cells MLE12 in Test Example 5;

[0054] Fig. 9 Toxicity experiments of 4-OI, M / C / C NPs, and M / C / OI / C NPs in mice in Test Example 5 (the concentration of each group was 20 mg / kg);

[0055] Fig.10The 7-day survival curve of the sepsis animal model (mice were induced by cecal perforation and ligation surgery, CLP) treated with 4-OI, M / C / C NPs, and M / C / OI / C NPs (the concentration of each group was 20 mg / kg) in Test Example 6;

[0056] Fig.11 is the concentration of TNF-α in peripheral blood at 12h after CLP mice were treated with 4-OI, M / C / C NPs, and M / C / OI / C NPs in Test Example 6;

[0057] Fig.12 is the concentration of IL-1β in peripheral blood at 12h after CLP mice were treated with 4-OI, M / C / C NPs, and M / C / OI / C NPs in Test Example 6;

[0058] Fig.13 is the concentration of IL-6 in peripheral blood at 12h after CLP mice were treated with 4-OI, M / C / C NPs, and M / C / OI / C NPs in Test Example 6;

[0059] Fig.14 In Test Example 6, after CLP mice were treated with 4-OI, M / C / C NPs, and M / C / OI / C NPs, at 24 h, the degree of damage to important organs was observed, with the lung tissue, which is the most vulnerable tissue in sepsis, as a representative, and the H&E staining, lung dry-wet ratio, and protein content in bronchoalveolar lavage fluid were observed;

[0060] Fig.15 In Test Example 7, the images were taken to observe the turbidity of the E. coli culture solution after 24 hours of treatment with 4-OI, M / C / C NPs, and M / C / OI / C NPs (the concentration of each group was 20 μg / mL);

[0061] Fig.16 In Test Example 7, after the E. coli solution was treated with 4-OI, M / C / C NPs, and M / C / OI / C NPs for 24 hours, the absorbance of the solution at 600 nm was measured, and the E. coli inhibition rate was calculated based on this;

[0062] Fig.17 The level of NETs marker (CitH3-DNA complex) in the peripheral blood of CLP mice at 24h after treatment with 4-OI, M / C / C NPs, and M / C / OI / C NPs in Test Example 8;

[0063] Fig.18The level of NETs markers (MPO-DNA complex) in the peripheral blood of CLP mice at 24 h after treatment with 4-OI, M / C / C NPs, and M / C / OI / C NPs in Test Example 8;

[0064] Fig.19 Immunofluorescence images of Ly6G and CitH3 in lung tissues of CLP mice treated with 4-OI, M / C / C NPs, and M / C / OI / C NPs at 24 h in Test Example 8;

[0065] Fig. 20 The present invention is a flow chart for preparing the antibacterial nanomedicine for treating sepsis. DETAILED DESCRIPTION

[0066] The technical solutions in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0067] The following examples illustrate the technical solution of the present invention by taking an antibacterial nanomedicine for treating sepsis as an example. The antibacterial nanomedicine for treating sepsis is a nanoparticle, which uses titanium carbide nanosheets (MXene) as a skeleton and self-assembles chitosan quaternary ammonium salt (QCS), 4-octylitaconic acid (4-OI) and neutrophil cell membrane (NMC) layer by layer. These ingredients have good biocompatibility and safety, are biodegradable, and the degradation products are non-toxic and do not cause inflammation. In addition, these ingredients also have the following characteristics:

[0068] Titanium carbide nanosheets: rich surface groups and easy to modify; large specific surface area, can be used as drug carriers; and have certain antibacterial ability.

[0069] Chitosan quaternary ammonium salt: a classic antibacterial macromolecule that can adsorb and fix bacteria and exhibit a powerful bactericidal effect.

[0070] 4-Octylitaconic acid: has slight antibacterial ability. The present invention found that it has a certain inhibitory effect on the production of NETs.

[0071] Neutrophil cell membrane: can capture bacteria, target neutrophils, and enhance the stability of nanoparticles in liquids.

[0072] The present invention provides the above-mentioned antibacterial nanomedicine for treating sepsis mainly to achieve two goals:

[0073] (1) Regulating the production of NETs: In view of the adverse effects of excessive NETs on patients with sepsis, the present invention hopes to appropriately inhibit the excessive production of NETs. To this end, the present invention uses the neutrophil cell membrane in the outermost layer of the antibacterial nanomedicine to enable it to target neutrophils. At the same time, the antibacterial nanomedicine is loaded with "4-octyl itaconic acid", which can effectively inhibit the excessive production of NETs.

[0074] (2) Elimination of bacteria: Simply regulating the production of NETs is not enough to cure sepsis. If the root cause of sepsis, severe bacterial infection, is not eliminated, sepsis still cannot be effectively treated. Considering that NETs themselves have a strong bactericidal effect, the present invention designed this antibacterial nanomedicine based on a bionic strategy. Its outermost layer is similar to NETs, ​​using neutrophil cell membranes (retaining their protein components and functions) to capture bacteria. At the same time, the present invention uses "titanium carbide nanosheets" to replace the DNA skeleton of NETs, ​​and uses "chitosan quaternary ammonium salt" and "4-octylitaconic acid" to simulate the bactericidal factors in NETs.

[0075] Therefore, the antibacterial nanomedicine for the treatment of sepsis is not only similar in structure to NETs and has the same strong bactericidal ability, but also has better biocompatibility and safety. It will not be destroyed and degraded by DNA enzymes, nor will it cause inflammatory factor storms and tissue damage. In addition, it can appropriately inhibit the excessive production of NETs. In general, the antibacterial nanomedicine for the treatment of sepsis can treat sepsis by regulating the production of NETs and inhibiting bacterial infection.

[0076] In addition, after degradation, the antibacterial nanomedicine mainly produces non-toxic and safe substances such as titanium ions, carbon dioxide and water, thereby effectively avoiding possible inflammatory reactions and tissue damage.

[0077] The following examples use MXene nanosheets as carriers to prepare antibacterial nanomedicines by layer-by-layer self-assembly. The entire process is green and environmentally friendly, does not involve complex instruments and equipment, and does not involve toxic and hazardous substances. The technical solution of the present invention is further explained by examples and comparative examples.

[0078] Example 1

[0079] (1) Preparation of MXene (Ti3C2) nanosheets:

[0080] 3.2g lithium fluoride (LiF) was dissolved in 40mL 9M hydrogen chloride (HCl) solution, and stirred at 500rpm for 2h at 40°C to ensure that LiF was dissolved to obtain 40mL LiF / HCl etching solution. 2g Ti3AlC2 powder was slowly added to 40mL LiF / HCl etching solution under ice bath conditions. Stir continuously at 500rpm for 48h at 45°C to obtain a stable MXene suspension. Wash repeatedly with deionized water (centrifuge at 3000rpm for 5min at room temperature, then resuspend with 50mL deionized water, centrifuge again, and repeat this process until the pH exceeds 6). Finally, centrifuge at 3000rpm for 30min at room temperature, take the precipitate and vacuum freeze-dry for 24h to obtain the product MXene (Ti3C2) nanosheets.

[0081] (2) Preparation of chitosan quaternary ammonium salt-modified MXene nanoparticles (named M / C NPs):

[0082] 50 mg of chitosan quaternary ammonium salt (QCS) powder was added to 50 mL of deionized water, stirred at 500 rpm for 2 h at room temperature to ensure that QCS was fully dissolved, and 50 mL of 1 mg / mL QCS solution was prepared. 20 mg of MXene nanosheets were added to 50 mL of 1 mg / mL QCS solution, stirred at 500 rpm for 24 h at room temperature to obtain a stable M / C NPs suspension. Washed repeatedly with deionized water 3 times (centrifuged at 3000 rpm for 5 min at room temperature, then resuspended with 50 mL of deionized water, centrifuged again, and repeated 3 times). Finally, centrifuged at 3000 rpm for 30 min at room temperature, and the precipitate was vacuum freeze-dried for 24 h to obtain the product M / C NPs.

[0083] (3) Preparation of composite nanoparticles loaded with 4-octylitaconic acid (named M / C / OI NPs):

[0084] 20 mg of 4-octylitaconic acid powder was added to 200 mL of deionized water, stirred at 500 rpm for 2 h at room temperature, and then ultrasonicated at 400 W for 10 min to prepare a stable 200 mL 0.1 mg / mL 4-OI suspension. 20 mg of M / C NPs was added to 200 mL of 0.1 mg / mL 4-OI suspension, stirred at 500 rpm for 24 h at room temperature, and then ultrasonicated at 400 W for 10 min to obtain a stable M / C / OI NPs suspension. The mixture was washed repeatedly with deionized water for 3 times (centrifuged at 3000 rpm for 5 min at room temperature, then resuspended with 50 mL of deionized water, centrifuged again, and repeated 3 times). Finally, the mixture was centrifuged at 3000 rpm for 30 min at room temperature, and the precipitate was freeze-dried in vacuum for 24 h to obtain the product M / C / OI NPs.

[0085] (4) Preparation of neutrophil cell membrane (NCM)-coated biomimetic nanoparticles (named M / C / OI / C NPs):

[0086] First, a neutrophil cell membrane (NCM) suspension was prepared: mouse peripheral blood neutrophils were separated and collected, the cells were resuspended in membrane protein extraction buffer, and ice bathed for 10 minutes. After that, the samples were repeatedly frozen and thawed in liquid nitrogen and room temperature three times. The resulting mixture was centrifuged (4°C, 700g, 10min), the supernatant was taken, and centrifuged again (4°C, 14000g, 30min). Finally, the supernatant was discarded and the precipitate was taken, which was the neutrophil cell membrane (NCM). 20mg NCM was resuspended in 20mL pre-cooled PBS buffer to prepare 20mL 1mg / mL NCM suspension. After that, 20mg M / C / OI NPs were added to the 20mg 1mg / mL NCM suspension, and ultrasonicated at 400W for 10min under ice bath conditions to obtain a stable M / C / OI / C NPs suspension. Centrifuged at 10000rpm for 30min at 4°C, the supernatant was discarded, and the precipitate was taken. The obtained precipitate was washed three times with pre-cooled PBS buffer (resuspended with 50 mL 4°C PBS buffer, and then centrifuged at 10000 rpm for 30 min at 4°C, repeated three times) to obtain biomimetic nanoparticles M / C / OI / C NPs. Finally, centrifuged at 3000 rpm for 30 min at 4°C, and the precipitate was vacuum freeze-dried for 24 h to obtain the product M / C / OI / CNPs.

[0087] Comparative Example 1

[0088] Preparation of MXene nanosheets: 3.2g lithium fluoride (LiF) was dissolved in 40mL 9M hydrogen chloride (HCl) solution, and stirred at 500rpm for 2h at 40°C to ensure that LiF was dissolved to obtain 40mL LiF / HCl etching solution. 2g Ti3AlC2 powder was slowly added to 40mL LiF / HCl etching solution under ice bath conditions. Stir continuously at 500rpm for 48h at 45°C to obtain a stable MXene suspension. Wash repeatedly with deionized water (centrifuge at 3000rpm for 5min at room temperature, then resuspend with 50mL deionized water, centrifuge again, and repeat this process until the pH exceeds 6). Finally, centrifuge at 3000rpm for 30min at room temperature, take the precipitate and vacuum freeze-dry for 24h to obtain the product MXene nanosheets.

[0089] Comparative Example 2

[0090] Preparation of chitosan quaternary ammonium salt modified MXene nanoparticles (named M / C NPs): 50 mg chitosan quaternary ammonium salt (QCS) powder was added to 50 mL deionized water, stirred at 500 rpm for 2 h at room temperature to ensure that QCS was fully dissolved, and 50 mL 1 mg / mL QCS solution was prepared. 20 mg MXene nanosheets were added to 50 mL 1 mg / mL QCS solution, stirred at 500 rpm for 24 h at room temperature to obtain a stable M / C NPs suspension. Wash repeatedly with deionized water 3 times (centrifuge at 3000 rpm for 5 min at room temperature, then resuspend with 50 mL deionized water, centrifuge again, and repeat 3 times). Finally, centrifuge at 3000 rpm for 30 min at room temperature, take the precipitate and vacuum freeze-dry for 24 h to obtain the product M / CNPs.

[0091] Comparative Example 3

[0092] Preparation of composite nanoparticles loaded with 4-octylitaconic acid (named M / C / OI NPs): 20 mg of 4-octylitaconic acid powder was added to 200 mL of deionized water, stirred at 500 rpm for 2 h at room temperature, and then ultrasonicated at 400 W for 10 min to prepare a stable 200 mL 0.1 mg / mL 4-OI suspension. 20 mg of M / C NPs was added to 200 mL of 0.1 mg / mL 4-OI suspension, stirred at 500 rpm for 24 h at room temperature, and then ultrasonicated at 400 W for 10 min to obtain a stable M / C / OI NPs suspension. Washed repeatedly with deionized water 3 times (centrifuged at 3000 rpm for 5 min at room temperature, then resuspended with 50 mL of deionized water, centrifuged again, and repeated 3 times). Finally, the mixture was centrifuged at 3000 rpm for 30 min at room temperature, and the precipitate was freeze-dried in vacuum for 24 h to obtain the product M / C / OI NPs.

[0093] Comparative Example 4

[0094] Preparation of neutrophil cell membrane-coated M / C NPs nanoparticles (named M / C / C NPs): First, prepare a neutrophil cell membrane (NCM) suspension: separate and collect mouse peripheral blood neutrophils, resuspend the cells with membrane protein extraction buffer, and ice bath for 10 minutes. After that, the sample was repeatedly frozen and thawed three times in liquid nitrogen and room temperature. The resulting mixture was centrifuged (4°C, 700g, 10min), the supernatant was taken, and centrifuged again (4°C, 14000g, 30min). Finally, the supernatant was discarded and the precipitate was taken, which was the neutrophil cell membrane (NCM). 20mg NCM was resuspended in 20mL pre-cooled PBS buffer to prepare 20mL 1mg / mL NCM suspension. After that, 20mg M / C NPs were added to the 20mg 1mg / mL NCM suspension, and ultrasonicated at a power of 400W for 10min under ice bath conditions to obtain a stable M / C / C NPs suspension. The mixture was centrifuged at 10000 rpm for 30 min at 4°C, the supernatant was discarded, and the precipitate was washed three times with pre-cooled PBS buffer (resuspended with 50 mL of 4°C PBS buffer, and then centrifuged at 10000 rpm for 30 min at 4°C, repeated three times), and the precipitate was vacuum freeze-dried for 24 h to obtain the product M / C / C NPs.

[0095] Test Example 1

[0096] Part of the obtained MXene nanosheets and M / C / OI / C nanoparticles were dissolved in ultrapure water, the middle liquid was taken and dropped onto a copper sheet, and the appearance of the nanoparticles was observed using a scanning electron microscope. It can be seen that the MXene nanosheets have a typical accordion-like two-dimensional multilayer morphology, which has the advantages of high specific surface area and high drug loading rate, while the outermost layer of the M / C / OI / C nanoparticles is evenly wrapped with a gray translucent cell membrane. The results are as follows Figure 1 shown.

[0097] Test Example 2

[0098] The M / C / OI / C NPs prepared in Example 1 and the MXene (Ti3C2) nanosheets, M / CNPs, and M / C / OI NPs prepared in Comparative Examples 1, 2, and 3 were respectively redissolved in ultrapure water and placed in a Malvern particle size analyzer to detect the particle size distribution and potential. The sample test was repeated three times and the average value was taken to measure the particle size, fractional index (PDI), and Zeta potential. The results are shown in FIG. Figure 2 , Figure 3 and Figure 4 As shown. The particle sizes of MXene nanosheets, M / C NPs, M / C / OI NPs and M / C / OI / C NPs were measured to be 250.533±5.962nm, 259.233±6.035nm, 260.533±5.138nm and 267.867±7.153nm, respectively. This gradual change in particle size provides some evidence for the successful preparation of each group of nanoparticles (see Figure 2 In order to further explore the stability of these nanoparticles, the present invention further monitored the particle size changes of M / C / OI / C NPs in ultrapure water for up to 7 days and calculated its dispersion index (PDI). The experimental results show that during these 7 days, the particle size and dispersion index of M / C / OI / C NPs remained stable with minimal fluctuations, which fully demonstrates that M / C / OI / C NPs have excellent stability in ultrapure water (see Figure 3 In addition, the present invention also measured the Zeta potential of MXene nanosheets, M / C NPs, M / C / OI NPs and M / C / OI / C NPs, which were recorded as -20.16±1.12, -4.62±0.69, -11.26±1.22 and -19.31±1.00 mV, respectively. These changes in Zeta potential also prove the successful preparation of each group of nanoparticles (see Figure 4). Through the above experimental results, the present invention can clearly see the significant differences in particle size and Zeta potential of each group of nanoparticles, which are strong evidence of the successful preparation of nanoparticles. At the same time, the long-term stability of M / C / OI / C NPs in ultrapure water also provides solid support for its potential in practical applications.

[0099] Test Example 3

[0100] The M / C / OI / C NPs prepared in Example 1 and the M / C / OI NPs prepared in Comparative Example 3, as well as the neutrophil cell membrane, were subjected to the following protein analysis: First, the samples were mixed with a protein loading buffer and heated at 95°C for 5-10 minutes. Subsequently, SDS polyacrylamide gel electrophoresis was performed and stained with Coomassie Brilliant Blue. The purpose of this step was to observe whether the protein components on the surface of the M / C / OI / C NPs were the same as those on the neutrophil cell membrane. The electrophoresis results are shown in Figure 2. Figure 5 As shown, it can be clearly seen that the M / C / OI NPs group did not show obvious protein components, while the protein bands displayed by the M / C / OI / C NPs group were completely consistent with the bands of the neutrophil cell membrane. This finding proves that the proteins on the surface of M / C / OI / C NPs are indeed derived from the neutrophil cell membrane, and their composition is exactly the same as the original cell membrane protein. Given that the main functions of the cell membrane are mostly achieved through the proteins on it, the above results also indirectly indicate that during the preparation process of M / C / OI / C NPs, the composition and function of the neutrophil cell membrane coated on the outermost layer are completely preserved without any damage.

[0101] Test Example 4

[0102] Nanoparticle hemolysis experiment: First, blood was collected from C57BL / 6 mice and centrifuged at 200g for 15 minutes to collect the red blood cell layer. Subsequently, the red blood cells were diluted 10 times with normal saline, and different nanoparticles were added and incubated at 37°C for 4 hours. After that, the sample was centrifuged at 3000rpm for 10 minutes, and the absorbance value of the supernatant was measured at a wavelength of 540nm using an enzyme reader to calculate the hemolysis rate. The experimental results are as follows: Figure 6 As shown. As a positive control, the present invention used ultrapure water mixed with blood, and the hemolysis rate was measured to be as high as 100% ± 1.46%. In contrast, the hemolysis rates of the 4-OI group, the M / C / C NPs group, and the M / C / OI / C NPs group were 6.70% ± 0.68%, 2.96% ± 0.68%, and 2.46% ± 0.42%, respectively (as shown in Figure 2). Figure 6The experimental results show that 4-OI can cause slight hemolysis, while M / C / C NPs show good biocompatibility and basically do not cause hemolysis. By encapsulating 4-OI inside M / C / C NPs, the M / C / OI / CNPs prepared by the present invention also show excellent biocompatibility and almost do not cause hemolysis.

[0103] Test Example 5

[0104] Evaluation of the biocompatibility of nanoparticles in vitro and in vivo: (1) The biocompatibility of different nanoparticles on mouse alveolar epithelial cells MLE12 (such as Figure 7 and Figure 8 (2) Nanoparticles were injected into mice via tail vein to observe their potential damage to major organs (such as Fig. 9 The results showed that compared with the blank control group "Con", 4-OI, M / C / CNPs and MC / OI / C NPs had no significant effect on the proliferation of MLE12 cells (see Figure 7 and Figure 8 ). This shows that all three materials show excellent cell compatibility and will not induce cytotoxic reactions even when in direct contact with cells. In order to further verify the biosafety of the nanoparticles, the present invention used H&E staining technology to perform pathological examinations on major organs. The experiment found that compared with the blank control group "Con", 4-OI, M / C / C NPs and MC / OI / C NPs did not cause obvious damage to key organs such as the heart, liver, spleen, lungs and kidneys (such as Fig. 9 In summary, 4-OI, M / C / C NPs and MC / OI / CNPs showed excellent biosafety in both in vitro cell experiments and in vivo animal experiments.

[0105] Test Example 6

[0106] The therapeutic effects of 4-OI, M / C / C NPs and M / C / OI / C NPs (the concentration of each group was 20 mg / kg, the first administration was injected via the tail vein immediately after model establishment, and then injected via the tail vein every 48 hours) on CLP mice are as follows: 7-day survival curve ( Fig.10 ) shows the survival status of mice in each group, and the levels of peripheral blood inflammatory factors ( Fig.11 , 12 , 13) reflected the degree of inflammatory response in each group of mice, and the degree of damage to important organs was evaluated by the pathological manifestations of lung tissue (results such as Fig.14 The experimental results showed that compared with the CLP group, the survival rates of mice in the 4-OI group and the M / C / C NPs group were improved, and the survival rate improvement effect of the M / C / OI / CNPs group was more significant (see Fig.10 ). Subsequently, the present invention further observed the systemic inflammatory response of each group of mice. The study found that compared with the CLP group, the levels of inflammatory factors (including TNF-α, IL-1β and IL-6) in the peripheral blood of mice in the 4-OI group and the M / C / C NPs group were reduced, and the level of inflammatory factors in the M / C / OI / CNPs group decreased more significantly (see Figure 11-13 This trend was also confirmed in the pathological H&E staining results of lung tissue (see Fig.14 ). Therefore, it can be concluded that 4-OI and M / C / C NPs have a certain therapeutic effect on septic mice, and when the two work synergistically (i.e., M / C / OI / C NPs), the therapeutic effect on septic mice is more significant.

[0107] Test Example 7

[0108] Study on the inhibitory effect of 4-OI, M / C / C NPs and M / C / OI / C NPs on Escherichia coli: 4-OI, M / C / C NPs and M / C / OI / C NPs with a concentration of 20 μg / mL were co-cultured with Escherichia coli culture for 24 hours. At 24 hours after the end of the culture, the present invention took optical photos of each group of culture solutions (such as Fig.15 The absorbance of the bacterial solution was measured at a wavelength of 600 nm (as shown in Fig.16 The experimental results showed that compared with the control group "Con", the turbidity of the bacterial solution in the 4-OI group only decreased slightly, while the turbidity of the bacterial solution in the M / C / C NPs group decreased significantly. It is worth mentioning that the bacterial solution in the M / C / OI / C NPs group was close to a clear state ( Fig.15 ). By calculating and analyzing the absorbance data, the present invention further verified the antibacterial effect of each group of materials, and the obtained antibacterial rate was consistent with the above observation results. In summary, 4-OI showed weak antibacterial ability, while M / C / C NPs and M / C / OI / C NPs showed excellent antibacterial effect.

[0109] Test Case 8

[0110] The regulatory effects of 4-OI, M / C / C NPs and M / C / OI / C NPs on neutrophil extracellular traps (NETs) in animal models of sepsis were studied. The levels of NETs markers (CitH3-DNA complex, MPO-DNA complex) in the peripheral blood of mice were detected by Elisa method. The specific results are as follows: Fig.17 and Fig.18At the same time, the fluorescence intensity and co-localization of Ly6G and CitH3 in lung tissue were observed using immunofluorescence confocal microscopy. The results are shown in Fig.19 The experimental results showed that compared with the CLP group, the levels of NETs markers in the peripheral blood of mice in the 4-OI group and the M / C / C NPs group showed a slight downward trend. Under the synergistic effect of the two (i.e., the M / C / OI / C NPs group), the levels of NETs markers in the peripheral blood of mice decreased significantly (as shown in Figure 2). Figure 17-18 In addition, the fluorescence intensity of CitH3 (one of the components of NETs) in mouse lung tissue also showed changes consistent with the above trend (as shown in Fig.19 As shown). Therefore, the present invention can conclude that 4-OI and M / C / C NPs have a certain regulatory effect on the production of NETs in sepsis. This regulatory effect is mainly attributed to the immunomodulatory activity of 4-OI and the antibacterial ability of M / C / C NPs. The biomimetic nanoparticles M / C / OI / C NPs obtained by combining the two show significant advantages in the treatment of sepsis: on the one hand, it can regulate the production of NETs and avoid the potential harm caused by excessive NETs; on the other hand, its structure and function are similar to NETs, ​​and it has excellent antibacterial effects, which helps the body to eliminate the cause of sepsis (i.e., a large number of invading pathogens, such as bacteria). In summary, M / C / OI / C NPs show good therapeutic effects in the treatment of sepsis.

[0111] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. An antibacterial nanomedicine, characterized in that: The antibacterial nano drug comprises: a nano skeleton, a NETs inhibitory drug and an antibacterial drug loaded on the nano skeleton, and a neutrophil cell membrane located at the outermost layer of the antibacterial nano drug; the nano skeleton is a multilayer nano sheet, and the multilayer nano sheet is a titanium carbide nano sheet; the NETs inhibitory drug is 4-octylitaconic acid; the antibacterial drug is a chitosan quaternary ammonium salt; the preparation method of the antibacterial nano drug comprises: making the nano skeleton contact with the antibacterial drug, the NETs inhibitory drug and the neutrophil cell membrane in a solution in turn, and performing layer-by-layer self-assembly.

2. An antibacterial composition, characterized in that: It comprises the antibacterial nanomedicine according to claim 1 as an active ingredient, and optionally one or more pharmaceutically acceptable excipients.

3. The antibacterial composition according to claim 2, characterized in that: The antibacterial composition does not contain other antibacterial active ingredients.

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