Membrane bionic nanomaterial for treating sepsis lung injury as well as preparation method and application of membrane bionic nanomaterial

Abstract

The invention relates to a membrane bionic nanomaterial for treating sepsis lung injury as well as a preparation method and application of the membrane bionic nanomaterial. The preparation method of the membrane bionic nanomaterial for treating the sepsis lung injury comprises the following steps: S1, preparing a targeted peptide DSPE-PEG-Tyr storage solution; s2, preparation of MM-Tyr is carried out; s3, preparing a solution; s4, loading of effective active ingredients; and S5, construction of the membrane bionic nano material Tyr-MM (at) PLGA / G + F. The membrane bionic nanomaterial Tyr-MM-coated PLG / G + F provided by the invention integrates sepsis lung injury treatment and inflammation response functions, can respond to an inflammation signal of a focus part of sepsis lung injury, is delivered to the focus part in a targeted manner, releases two effective active components, exerts synergistic antibacterial, anti-inflammatory and immunomodulatory effects, and has a good application prospect in treatment of sepsis lung injury. The key link of the pathologic process of sepsis is effectively intervened, and the huge potential of preventing and treating sepsis lung injury is shown.

Description

Technical Field

[0001] The present invention belongs to the field of biomaterials, and in particular relates to a membrane biomimetic nanomaterial for treating septic lung injury, and a preparation method and application thereof. Background Art

[0002] Sepsis is a systemic inflammatory response syndrome (SIRS), characterized by the massive secretion of proinflammatory cytokines and the subsequent multi-organ failure. Acute lung injury (ALI) is a severe complication of SIRS and carries a high mortality rate. Sepsis-induced lung injury (SILI) is a common clinical critical illness in intensive care units. The typical pathological manifestations of SILI are disruption of the alveolar-capillary barrier and increased vascular permeability, leading to refractory hypoxemia and high morbidity and mortality.

[0003] Currently, there are no specific medications or effective treatments for sepsis-induced acute lung injury (ALI). The current clinical treatment process for patients diagnosed with SLI is as follows: First, early resuscitation is initiated, primarily to maintain basic vital signs. Next, anti-infective therapy is initiated. Physicians select appropriate antibiotics, such as penicillins, cephalosporins, and quinolones, based on the results of pathogen testing. Broad-spectrum antibiotics are often used to quickly control infection. However, the increasing prevalence of drug-resistant bacteria in recent years has led to a bottleneck in antibiotic therapy, which can also lead to dysbiosis and an increased risk of secondary infection. Finally, supportive and symptomatic treatments are used to further manage SLI patients, primarily including mechanical ventilation, fluid management, immunomodulatory therapy, anticoagulation, and microcirculatory improvement. Therefore, the development of efficient, safe, and targeted drug delivery systems for SLI, such as the use of nanotechnology to achieve precise targeting and controlled release, has become a research hotspot and a pressing need. By optimizing the drug delivery strategy, increasing the drug distribution concentration and retention time in the lungs, and reducing systemic side effects, it is expected to bring about a revolutionary breakthrough in the treatment of septic lung injury.

[0004] In recent years, cell membrane biomimetic nanotechnology has become a highly promising therapeutic platform. This technology fuses natural cell membranes to the surface of artificially synthesized nanoparticles (NPs), allowing these nanoparticles to inherit the specific biological characteristics and functions of the source cells, such as prolonging blood circulation time and the ability to precisely target disease-related tissues. Macrophage membrane-coated nanoparticles (MCM-NPs) are macrophage membranes coated on different types of nanocarriers, giving these nanoparticles a series of unique biological functions and characteristics. In previous research explorations, macrophage membrane-coated nanoparticles have demonstrated efficient targeted delivery performance and significant therapeutic effects on a variety of inflammatory diseases. In addition, a large number of studies have further revealed the key role of macrophage membranes in the treatment of septic lung injury. For example, in sepsis, endotoxin (also known as lipopolysaccharide, LPS), as a product released by bacteria during cell division, apoptosis or antibiotic intervention, is recognized as pathogen-associated molecular patterns (PAMPs) by sentinel immune cells, especially monocytes and macrophages, thereby triggering a complex immune response. Macrophage membrane-mimicking nanomaterials also acquire the ability to bind to endotoxin by simulating and inheriting the protein composition of the macrophage membrane surface. This property helps to reduce the downstream inflammatory response caused by endotoxin.

[0005] Existing studies have shown that proteins such as integrin α4β1 are also expressed on the surface of macrophage membranes. These proteins can "home" to areas of atherosclerotic lesions and actively bind specifically to vascular cell adhesion molecule-1 (VCAM-1), which is highly expressed on inflamed endothelial cells. Given that VCAM-1 also plays an important role in the pathological process of lung injury, this discovery provides new opportunities and strategies for the application of macrophage membrane coating nanotechnology in the treatment of lung injury. Therefore, innovative therapies based on macrophage membrane coating nanotechnology, through precise targeting and biological function simulation, are expected to open up new avenues for the treatment of complex diseases such as inflammatory diseases and septic lung injury. Summary of the Invention

[0006] In response to the defects of the existing technology, the present invention provides a membrane biomimetic nanomaterial for the treatment of septic lung injury, its preparation method and application. The membrane biomimetic nanomaterial Tyr-MM@PLGA / G+F can effectively intervene in the key links of the pathological process of sepsis through multiple mechanisms such as antibacterial, anti-inflammatory, targeted delivery of inflammation and immune regulation, showing great potential for preventing and treating septic lung injury.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The first object of the present invention is to provide a method for preparing a membrane biomimetic nanomaterial for treating septic lung injury, comprising the following steps:

[0009] S1. Preparation of Targeting Peptide DSPE-PEG-Tyr Storage Solution:

[0010] The targeting peptide DSPE-PEG-Tyr powder was added to a certain amount of PMSF to prepare a 0.1-10 mg / mL targeting peptide DSPE-PEG-Tyr storage solution for later use;

[0011] Preparation of S2, MM-Tyr:

[0012] The target peptide DSPE-PEG-Tyr stock solution prepared in step S1 was added to a 0.5-2 mg / mL PBS solution of macrophage membrane MM and incubated at 4°C for 12 hours to finally prepare MM-Tyr;

[0013] S3. Preparation of solution:

[0014] Dissolve geraniol in chloroform to prepare a geraniol solution with a final concentration of 5-10 μg / mL, and set aside; dissolve the antimicrobial peptide FK13-a1 in water to prepare an antimicrobial peptide FK13-a1 aqueous solution with a final concentration of 12-16 μg / mL, and set aside; prepare a PVA-CS storage solution containing 0.8-1.2% polyvinyl alcohol (PVA) and 0.1-0.3% chitosan (CS), and set aside;

[0015] S4. Loading of active ingredients:

[0016] The antimicrobial peptide FK13-a1 aqueous solution prepared in step S3 is added to the geraniol solution and subjected to a first sonication to prepare an O / W nanoemulsion; the O / W nanoemulsion is then added to the PVA-CS storage solution prepared in step S3 and subjected to a second sonication to prepare a nanoemulsion PLGA / G+F;

[0017] S5. Construction of membrane biomimetic nanomaterial Tyr-MM@PLGA / G+F:

[0018] The MM-Tyr prepared in step S2 and the nanoemulsion PLGA / G+F prepared in step S3 were gently mixed at a mass ratio of 1:(0.2-0.8) and incubated overnight at 2-6°C; the solution was extruded through a PC membrane with a pore size of 200 nm and collected to prepare a membrane biomimetic nanomaterial Tyr-MM@PLGA / G+F.

[0019] Preferably, the targeting peptide DSPE-PEG-Tyr powder in step S1 is a targeting peptide DSPE-PEG-Tyr powder with a purity greater than 95% HPLC purity.

[0020] Preferably, the mouse mononuclear macrophage leukemia cell-derived biofilm in step S2 is obtained by culturing Raw264.7 cells, collecting, and extracting the cell membrane of the Raw264.7 cells.

[0021] Preferably, the culturing of Raw264.7 cells comprises: culturing the Raw264.7 cells in a 35-45°C, 2-8% CO2 environment using DMEM complete medium, discarding the original medium when the cell density reaches about 75-80%, washing 2-3 times with PBS buffer to completely remove the cells, collecting the cell suspension, and temporarily storing it in a -80-100°C low-temperature refrigerator for future use.

[0022] Preferably, the amino acid sequence of the antimicrobial peptide FK13-a1 in step S3 is shown as SEQ.ID.NO.1.

[0023] Preferably, the conditions of the first ultrasound in step S4 are 2 to 4 seconds each time, 1 to 3 seconds interval, and an amplitude of 20 to 40%.

[0024] Preferably, the second ultrasound in step S4 is performed for 2 to 4 seconds at a time, with an interval of 1 to 3 seconds and an amplitude of 20 to 40%.

[0025] The second object of the present invention is to provide a membrane biomimetic nanomaterial prepared by the above preparation method.

[0026] Another object of the present invention is to provide a use of the membrane biomimetic nanomaterial as described above, wherein the membrane biomimetic nanomaterial is used to prepare a drug for treating septic lung injury.

[0027] The design principle of the present invention:

[0028] Targeting peptide DSPE-PEG-Tyrs: This is an advanced nanocarrier system that combines a phospholipid (DSPE), polyethylene glycol (PEG), and tyrosine (Tyr) or its derivative peptides. Through a specific chemical connection, this structure combines the membrane affinity of phospholipids, the hydrophilicity of PEG, and the potential targeting of Tyr or its derivative peptides. It can precisely deliver drugs or other bioactive substances to specific cells or tissues, achieving targeted therapy, and has broad application potential in biomedical research and drug delivery.

[0029] Geraniol, also known as geraniol, is a colorless to yellow oily liquid with a mild rose aroma. It is a major component of essential oils such as rose oil and citronella oil. It is widely used in daily fragrances, food flavors, and pharmaceuticals. Geraniol is a natural monoterpene alcohol with multi-target pharmacological effects.

[0030] Antimicrobial peptide FK13-a1: A short 13-amino acid α-helical peptide, it is derived from the optimized and modified amino acid sequence of the human antimicrobial peptide LL-37. It exhibits broad-spectrum antimicrobial activity, effectively inhibiting the growth of a variety of microorganisms, including Gram-positive and Gram-negative bacteria, fungi, and yeasts, including Staphylococcus aureus, Propionibacterium acnes, and Pseudomonas aeruginosa. Furthermore, antimicrobial peptide FK13-a1 may also promote wound healing, repair the skin barrier, and enhance immunity. In the medical field, it is considered a promising new antimicrobial drug ingredient for the development of wound dressings, skin repair products, and more.

[0031] Macrophage membrane (MM): It is a lipid bilayer structure on the surface of macrophages, mainly composed of phospholipids, proteins and carbohydrates. It is rich in membrane proteins, lipids and carbohydrate molecules. It is responsible for maintaining the morphology of cells, supporting the interaction between cells and the external environment, and plays a vital role in immune response.

[0032] The schematic diagram of the material construction of the membrane biomimetic nanomaterial Tyr-MM@PLGA / G+F provided by the present invention is as follows Figure 1 As shown, first, the targeting peptide DSPE-PEG-Tyr was complexed with a biomembrane derived from mouse mononuclear macrophage leukemia cells. The hydrophobic segment DSPE of the targeting peptide DSPE-PEG-Tyr was used to insert Tyr into the cell membrane, forming the targeting moiety MM-Tyr, thereby enhancing the material's specificity for the lesion site. Second, PLGA was used as a liposome to encapsulate geraniol and the antimicrobial peptide FK13-a1 via a water-in-oil-in-water technique, forming a nanoemulsion PLGA / G+F as the main active ingredient. Finally, the targeting moiety MM-Tyr was complexed with the nanoemulsion PLGA / G+F to construct the membrane-forming biomimetic nanomaterial Tyr-MM@PLGA / G+F.

[0033] The process of septic lung inflammation involves the interaction of multiple cells and molecules, including the activation of inflammatory cells, the release of cytokines, and intercellular adhesion. In this process, there is a large infiltration of inflammatory cells such as neutrophils and macrophages in both acute and chronic inflammatory tissues, which secrete excessive amounts of myeloperoxidase (MPO) and reactive oxygen species (ROS). In this process, the membrane biomimetic nanomaterial Tyr-MM@PLGA / G+F provided by the present invention is recruited to the site of inflammation in the presence of the targeting moiety MM-Tyr. In addition, tyramine (Tyr) modification can significantly enhance the targeted aggregation of nanoparticles in the local area of inflammation. This is mainly because the phenolic group can form free radicals under the action of excessive MPO and ROS in the pathological microenvironment, and further form polymers, which promotes the in situ cross-linking and tissue anchoring of the nanoparticles. The entire nanomaterial is brought into the lesion and released.

[0034] Poly(lactic-co-glycolic acid) (PLGA) is a biodegradable polymer material. It is composed of two monomers, lactic acid (LA) and glycolide (GA), through random or block copolymerization. PLGA degrades primarily in the body through non-enzymatic hydrolysis. The ester bonds in its molecular chain undergo hydrolysis and cleavage with water molecules in the body fluid environment, gradually breaking down into lactic acid (LA) and glycolic acid (GA) monomers. Both monomers are normal intermediates in human metabolism and are ultimately metabolized into carbon dioxide and water through the tricarboxylic acid cycle or excreted through the kidneys. Therefore, under inflammatory conditions, PLGA degrades, exposing geraniol and the antimicrobial peptide FK13-a1. Geraniol and the antimicrobial peptide FK13-a1 act together to exert anti-inflammatory and antibacterial effects in various conditions, including inflammation caused by septic lung injury, bacterial infection, and endothelial damage. Thus, the membrane biomimetic nanomaterial Tyr-MM@PLGA / G+F constructed in this paper possesses multiple functions.

[0035] Furthermore, the present invention conducted in-depth research on the therapeutic effects of the membrane biomimetic nanomaterial Tyr-MM@PLGA / G+F by constructing an animal model of septic lung injury. The results showed that the material effectively intervened in key aspects of sepsis pathogenesis through multiple mechanisms, including antibacterial, anti-inflammatory, targeted inflammation delivery, and immunomodulatory mechanisms, significantly improving lung injury.

[0036] Therefore, the present invention not only provides a new treatment method in the field of nanomedicine, but also provides a new option for the clinical treatment of septic lung injury, which has certain scientific significance and clinical application value.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] (1) The present invention innovatively provides a membrane biomimetic nanomaterial Tyr-MM@PLGA / G+F that can specifically identify sepsis. The obtained membrane biomimetic nanomaterial Tyr-MM@PLGA / G+F was used to study septic lung injury. It was found that the membrane biomimetic nanomaterial Tyr-MM@PLGA / G+F provided by the present invention has significant targeted antibacterial and anti-inflammatory effects in the treatment of septic lung injury.

[0039] (2) The present invention encapsulates geraniol and antimicrobial peptide FK13-a1 through the water-in-oil-in-water technology to form a nanoemulsion PLGA / G+F as the main active ingredient. The targeting peptide DSPE-PEG-Tyr is compounded with a biomembrane derived from mouse mononuclear macrophage leukemia cells, and Tyr is inserted into the cell membrane using the hydrophobic segment DSPE of the targeting peptide DSPE-PEG-Tyr to form a targeting portion MM-Tyr, thereby preparing a membrane biomimetic nanomaterial with a targeted therapeutic effect on septic lung injury. The preparation method is simple and easy to operate and is suitable for industrial production.

[0040] (3) The present invention uses geraniol and antimicrobial peptide FK13-a1 to treat septic lung injury. The synergistic effect of geraniol and antimicrobial peptide FK13-a1 achieves dual antibacterial and anti-inflammatory effects, providing a new idea for the combined treatment of septic lung injury, that is, a two-pronged and mutually reinforcing treatment idea of antibacterial and anti-inflammatory.

[0041] (4) The present invention establishes a cell-targeted drug delivery system for septic lung injury, achieving specific intervention on septic lung injury tissue and avoiding the impact on normal cells, which brings good news to sepsis patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 Diagram of the construction process of membrane biomimetic nanomaterial Tyr-MM@PLGA / G+F;

[0043] Figure 2 TEM image of membrane biomimetic nanomaterial Tyr-MM@PLGA / G+F;

[0044] Figure 3 This is the drug loading rate curve of the antimicrobial peptide FK13-a1 in the membrane biomimetic nanomaterial Tyr-MM@PLGA / G+F;

[0045] Figure 4 This is the geraniol drug loading rate curve of the membrane biomimetic nanomaterial Tyr-MM@PLGA / G+F;

[0046] Figure 5 This is the SEM image of the in vitro inflammatory response of the membrane biomimetic nanomaterial Tyr-MM@PLGA / G+F;

[0047] Figure 6 This is a diagram showing the in vitro anti-inflammatory effect of membrane biomimetic nanomaterial Tyr-MM@PLGA / G+F;

[0048] Figure 7 To evaluate the antibacterial effect of geraniol combined with antimicrobial peptide FK13-a1 in vitro;

[0049] Figure 8This is a diagram showing the in vivo lung therapeutic effect of membrane biomimetic nanomaterial Tyr-MM@PLGA / G+F;

[0050] Figure 9 Diagram of the in vivo antibacterial effect of membrane biomimetic nanomaterial Tyr-MM@PLGA / G+F;

[0051] Figure 10 Diagram of the in vivo anti-inflammatory effect of membrane biomimetic nanomaterial Tyr-MM@PLGA / G+F;

[0052] Figure 11 Diagram of the in vivo immunomodulatory effect of membrane biomimetic nanomaterial Tyr-MM@PLGA / G+F; DETAILED DESCRIPTION

[0053] The present invention will be further explained below with reference to specific examples. However, it should be noted that the following examples are intended only to illustrate the present invention and are not intended to limit the present invention. All technical solutions that are identical or similar to the present invention are within the scope of protection of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.

[0054] 1. Reagents and biological materials

[0055] The mouse mononuclear macrophage leukemia cells (Raw264.7 cells), human renal cortical proximal tubule epithelial cells HK2, and human alveolar basal epithelial cells A549 were purchased from the China Center for Type Culture Collection, Wuhan University.

[0056] The methicillin-resistant Staphylococcus aureus (MRSA) was deposited in Guangdong Provincial Microbiological Culture Collection Center with the deposit number GDMCC No: 1.644, and Pseudomonas aeruginosa (PA, ATCC 15442) was purchased from Guangdong Provincial Microbiological Culture Collection Center;

[0057] The human normal hepatocytes LO2 were purchased from Wuhan Punosai Life Science Technology Co., Ltd.

[0058] The human bronchial epithelial cells BEAS-2B were purchased from Wuhan Saiweier Biotechnology Co., Ltd.

[0059] The antimicrobial peptide FK13-a1 was synthesized by Beijing Zhongke Yaguang Biotechnology Co., Ltd.

[0060] The targeting peptide DSPE-PEG-Tyr is synthesized by Xi'an Ruixi Biotechnology Co., Ltd. and is a HPLC-pure targeting peptide DSPE-PEG-Tyr powder with a purity of >95%;

[0061] Unless otherwise specified, the culture medium is the commonly used culture medium in the laboratory, DMEM and DMEM / F12 medium;

[0062] The fetal bovine serum (FBS) was purchased from Gibco, USA;

[0063] The male BALB / c mice (18-22 g) were purchased from Guangdong Medical Experimental Animal Center.

[0064] 2. Instruments and equipment

[0065] Cell disruptor Sonics&Materials.

[0066] 3. Extraction and purification of cell membranes from mouse mononuclear macrophage leukemia cells (Raw264.7 cells)

[0067] (1) Cultivation of RAW264.7 cells: Raw264.7 cells were cultured in DMEM complete medium (10% fetal bovine serum (Gibco) + 1% (v / v) penicillin / streptomycin (P / S, Gibco)) at 37°C in a 5% CO2 environment. When the cell density reached about 80%, the original medium was discarded and the cells were rinsed three times with PBS buffer to completely detach the cells. The cell suspension was collected and temporarily stored in a -80°C low-temperature refrigerator.

[0068] (2) Extraction of macrophage membranes: Cell membranes were extracted using a cell membrane protein and plasma protein extraction kit (purchased from Bio-Tech Biotechnology Co., Ltd., model P0033). The specific steps were as follows: centrifuge at 700 × g for 10 minutes to collect the cell pellet and take the supernatant. Melt the membrane protein extraction buffer A at room temperature and quickly store it on ice. Dissolve PMSF in buffer A at 4°C to a final concentration of 1 mM. After obtaining the extraction working solution, quickly disperse the cells in it and treat it in an ice bath for 12 minutes.

[0069] Cell disruption: Collect the pretreated cell suspension in a pre-cooled glass cell homogenizer and homogenize slowly and evenly at 4°C for 35 times to fully lyse the cells under physical action.

[0070] (3) Isolation and purification: Collect the product from the previous step in a 2 mL centrifuge tube and centrifuge at 700 g for 10 min at 4°C. Carefully collect the supernatant (it is better to take a small amount of the supernatant and avoid touching the precipitate). Centrifuge at 14,000 g for 30 min at 4°C. Collect the precipitate at the bottom of the tube, which is the macrophage membrane fragment (MM). Resuspend the membrane fragment in ultrapure water, freeze-dry, and store in a -80°C refrigerator.

[0071] Example 1 Preparation method of membrane biomimetic nanomaterials for treating septic lung injury of the present invention

[0072] The method for preparing a membrane biomimetic nanomaterial for treating septic lung injury of the present invention comprises the following steps:

[0073] S1. Preparation of Targeting Peptide DSPE-PEG-Tyr Storage Solution

[0074] Add 1 mg of targeting peptide DSPE-PEG-Tyr powder into 500 μL of PMSF to prepare the targeting peptide DSPE-PEG-Tyr storage solution for later use;

[0075] Preparation of S2 and MM-Tyr

[0076] The DSPE-PEG-Tyr stock solution prepared in step S1 was added to 9.5 mL of PBS solution containing 10 mg of macrophage membrane MM, and incubated at 4°C for 12 hours to finally prepare MM-Tyr;

[0077] S3. Preparation of solution

[0078] Dissolve geraniol in 3 mL of chloroform to prepare a geraniol solution with a final concentration of 64.1 μg / mL for later use;

[0079] The antimicrobial peptide FK13-a1 was dissolved in water to prepare an antimicrobial peptide FK13-a1 aqueous solution with a final concentration of 71.7 μg / mL, and the solution was set aside;

[0080] 1 g of polyvinyl alcohol (PVA) and 0.2 g of chitosan (CS) powder were added to 100 mL of sterile PBS solution and the pH was adjusted with acetic acid to dissolve the chitosan to prepare a 1% (m / v) PVA-CS stock solution for later use.

[0081] S4. Loading of active ingredients

[0082] The antimicrobial peptide FK13-a1 aqueous solution prepared in step S3 was added to the geraniol solution and sonicated for a first time for 3 seconds each time, 2 seconds interval, and 30% amplitude to prepare an O / W nanoemulsion; the O / W nanoemulsion was then added to the PVA-CS storage solution and sonicated for a second time for 3 seconds each time, 2 seconds interval, and 30% amplitude to prepare a nanoemulsion PLGA / G+F;

[0083] S5. Construction of membrane biomimetic nanomaterial Tyr-MM@PLGA / G+F

[0084] The MM-Tyr prepared in step S2 and the nanoemulsion PLGA / G+F prepared in step S3 were gently mixed at a mass ratio of 1:0.5 and incubated overnight at 4°C; the solution was extruded through a PC membrane with a pore size of 200 nm and collected to prepare the membrane biomimetic nanomaterial Tyr-MM@PLGA / G+F.

[0085] Example 2 Preparation of PLGA / G+F and MM@PLGA / G+F

[0086] PLGA / G+F and MM@PLGA / G+F were prepared using the same preparation method as that of the membrane biomimetic nanomaterial Tyr-MM@PLGA / G+F in Example 1:

[0087] (1) Preparation of PLGA / G+F:

[0088] The nanoemulsion PLGA / G+F was prepared using the method of step S4 in Example 1.

[0089] (2) Preparation of MM@PLGA / G+F:

[0090] The macrophage membrane MM at a mass ratio of 1:0.5 was gently mixed with the nanoemulsion PLGA / G+F prepared in step S4 of Example 1 and incubated overnight at 4°C; the solution was extruded through a PC membrane with a pore size of 200 nm and collected to prepare the membrane biomimetic nanomaterial MM@PLGA / G+F.

[0091] Experiment 1 TEM observation of macrophage membrane complex structure

[0092] The PLGA / G+F, MM@PLGA / G+F and Tyr-MM@PLGA / G+F prepared in Example 1 and Example 2 were photographed using a transmission electron microscope (Hitachi High-Technologies Co., Ltd., model HT7700).

[0093] The experimental results are as follows Figure 2 As shown, there are dense spheres connected to the macrophage membrane, indicating that the construction is successful, which is the membrane biomimetic nanomaterial Tyr-MM@PLGA / G+F.

[0094] Experiment 2 Evaluation of drug loading efficiency of membrane biomimetic nanomaterials

[0095] In order to accurately evaluate the content of antimicrobial peptide FK13-a1 and geraniol in the membrane biomimetic nanomaterial, the effective active ingredient was loaded using an emulsification method in combination with step S4 of Example 1 (the antimicrobial peptide FK13-a1 aqueous solution was added to the geraniol solution and sonicated. The sonication duration was set to 2 minutes, the single sonication time was 3 seconds, the interval time was 2 seconds, and the amplitude was adjusted to 30%; the O / W nanoemulsion was then added to the PVA-CS storage solution and sonicated a second time under the same sonication conditions to prepare the nanoemulsion PLGA / G+F); the supernatant was collected by centrifugation at 12000 r / min for 10 minutes for drug loading rate calculation. The initial concentrations of antimicrobial peptide FK13-a1 and geraniol were 100 μg / mL, and the supernatant was finally diluted 5 times for absorbance testing.

[0096] The concentrations of antimicrobial peptide FK13-a1 standard samples were set at 8, 10, 12, 14, 16, 18, and 20 μg / mL, and a standard curve was prepared for these concentrations using an ultraviolet spectrophotometer at 280 nm.

[0097] The concentrations of geraniol standard samples were set to 1.058, 2.11, 3.164, 4.219, 5.174, 6.329, 8.438, 9.493, and 10.548 μg / mL, and standard curves were drawn at 208.9 nm.

[0098] A linear regression was performed with absorbance (OD value) as the ordinate and concentration (C, μg / mL) as the abscissa to draw a standard curve. The drug loading rate was determined using the standard curve method, where the drug loading rate was calculated as follows:

[0099] Drug loading rate (%) = (a*OD+b)×100% / C

[0100] Wherein, a and b are the slope and intercept of the drug loading standard curve, OD is 280 nm for FK13-a1 and 208.9 nm for geraniol, and C is the initial concentration of the antimicrobial peptide FK13-a1 or geraniol during drug loading in step S4 of Example 1.

[0101] The experimental results are as follows Figure 3 and Figure 4 As shown, among them, antimicrobial peptide a=0.00546, b=-0.00102; geraniol a=0.03865, b=0.11868), it can be seen that the drug loading rate of the membrane biomimetic nanomaterial Tyr-MM@PLGA / G+F provided by the present invention is 26% for antimicrobial peptide FK13-a1 and 78% for geraniol, which proves that the antimicrobial peptide FK13-a1 and geraniol are successfully loaded into the nanomaterial.

[0102] Experimental three-membrane biomimetic nanomaterial Tyr-MM@PLGA / G+F inflammatory responsiveness

[0103] Sepsis lung inflammation is a complex biological process involving the interaction of multiple inflammatory cells and molecules, characterized by a large infiltration of inflammatory cells such as neutrophils and macrophages, accompanied by excessive secretion of myeloperoxidase (MPO) and reactive oxygen species (ROS). Based on this, the present invention constructed a model simulating sepsis lung inflammation in vitro for experiment. The model construction method is shown in the literature [Nie Q, Li C, Wang Y, Hu Y, Pu W, Zhang Q, Cai J, Lin Y, Li G, Wang C, Li L, Dou Y, Zhang J. Pathologically triggered in situ aggregation of nanoparticles for inflammation-targeting amplification and therapeutic potentiation [J]. Acta Pharmaceutica Sinica B, 2023, 13 (1): 390-409. DOI: 10.1016 / j.apsb.2022.07.013.].

[0104] The inflammatory response experiment specifically includes the following steps:

[0105] The Tyr-MM@PLGA / G+F prepared in Example 1 of the present invention was tested for its inflammatory response. A blank PLGA, PLGA / G+F prepared in Example 2, and MM@PLGA / G+F were used as controls. The membranes were incubated in a mixed solution of 1 mmol / L H2O2 and 10 μg / mL MPO for 8 hours and observed using a scanning electron microscope (ZEISS GeminiSEM 300, Germany). The experiments demonstrated that the Tyr-MM@PLGA / G+F membrane biomimetic nanomaterial exhibits inflammatory response properties.

[0106] The experimental results are as follows Figure 5 As shown by Figure 5It can be seen that the nanomaterials can be clearly observed to aggregate under the inflammatory environment in the image, which has a targeted effect on inflammation. It can be seen that the membrane biomimetic nanomaterial Tyr-MM@PLGA / G+F provided by the present invention can be successfully recruited and anchored to the simulated inflammation site, realizing the release of the entire nanomaterial into the lesion, thereby providing a new strategy for the treatment of septic lung inflammation. The possible reason is that: the membrane biomimetic nanomaterial Tyr-MM@PLGA / G+F provided by the present invention will be recruited to the inflammation site in the presence of the targeting portion MM-Tyr, and in addition, tyramine (Tyr) modification can significantly enhance the targeted aggregation effect of nanoparticles in the local inflammation. This is mainly due to the fact that the phenolic group can form free radicals under the action of excessive MPO and ROS in the pathological microenvironment, and further form polymers, which promotes the in-situ crosslinking and tissue anchoring of the nanoparticles, and brings the entire nanomaterial into the lesion for release.

[0107] Experiment 4 Evaluation of the anti-inflammatory effect of membrane biomimetic nanomaterial Tyr-MM@PLGA / G+F in vitro

[0108] Reducing the inflammatory response in the body during the inflammatory cytokine storm is the key to the treatment of sepsis. It can effectively reduce mortality and reduce multi-organ damage caused by inflammation during infection. Therefore, this experiment used an in vitro inflammatory cell model to evaluate the anti-inflammatory effect of the material, and the inflammatory effect was measured by ELISA kit. The laboratory routinely cultured mouse mononuclear macrophage Raw264.7 cells, collected RAW264.7 cells in the logarithmic growth phase, that is, when the cells grew to 70-80% of the culture flask, discarded the culture medium, added sterile PBS and gently shook, repeated 3 times, discarded PBS, added culture medium (90% DMEM + 10% FBS), and used a pipette to aspirate the culture medium and blow the bottom of the culture flask to suspend the cells, counted the cells, and added culture medium to make the cell density 5×10 5 cells / well l, 3 mL per well was inoculated into a 6-well culture plate and cultured overnight in a 5% CO2 incubator.

[0109] The experiment was divided into a normal group (Normal group), an LPS+ATP group (Model group), an experimental group (the experimental group was divided into 7 groups: PLGA / G+F group, MM@PLGA / G+F group and Tyr-MM@PLGA / G+F group and a blank group (used as a blank control, no cells were added, and culture medium was added to offset the increase in OD value caused by the culture medium). The working concentration of LPS was set to 1 μg / mL, and the working concentration of ATP was 5 mmol / L. The normal group and the blank group were added with culture medium (without LPS and ATP, normal culture medium), and the LPS+ATP group (Model group) and the experimental group were added with LPS+ and placed in an incubator (37°C, 5% After culturing in 5% CO2 for 24 hours, the culture medium was discarded and the cells were washed three times with PBS. 1 mL of culture medium (normal culture medium) was added to the normal group and blank group, 200 μl of culture medium without drug was added to the LPS+ATP group (Model group), and 200 μl of culture medium without drug was added to the experimental group, respectively. The synthetic concentration was combined with the drug loading rate to achieve an in vitro therapeutic concentration of 50 μg / mL and 18.65 μg / mL. The drug concentrations in each group were 18.65 μg / mL for FK13-a1 and 50 ug / mL for geraniol. After incubation for 24 hours, the cell supernatant was collected and TNF-α, IL-1β and IL-6 were detected using the ELISA kit (purchased from ELISA Biotechnology Co., Ltd.) according to the instructions.

[0110] The experimental results are as follows Figure 6 As shown, the results show that the membrane biomimetic nanomaterial Tyr-MM@PLGA / G+F provided by the present invention can greatly reduce the content of LPS-induced inflammatory factors.

[0111] Evaluation of the antibacterial effect of spice leaf alcohol combined with antimicrobial peptide FK13-a1 in vitro

[0112] In order to evaluate the significant antibacterial effect of geraniol combined with the antimicrobial peptide FK13-a1, another common plant essential oil, eugenol (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., model E427189-1ml), was introduced for comparative experiments. Eugenol and geraniol are both components of plant essential oils and have similar pharmacological effects such as antidepressant and antianxiety activities. Both have broad-spectrum antimicrobial activity (effective against bacteria and fungi) and similar anti-inflammatory mechanisms. The specific implementation method of the comparative experiment is as follows:

[0113] After thawing the glycerol bacteria of Pseudomonas aeruginosa and Methicillin-resistant Staphylococcus aureus, inoculate them on an LB agar plate by the three-zone streaking method, and incubate at 37 °C for 8-10 h to form single colonies of appropriate size. Pipette 12 mL of LB liquid medium into a 20 mL screw-cap shaking tube that has been sterilized by high pressure. Pick a single colony and transfer it into the LB liquid medium, then place it in a constant temperature shaker at 37 °C and culture at 180 rpm for 12 h to obtain the original bacterial solution. Prepare a 96-well plate, add the geraniol and the antibacterial peptide FK13-a1 solution to the 96-well plate according to the serial dilution method, 100 μL per well. Add the diluted bacterial solution to the 96-well plate, 100 μL per well. Incubate at a constant temperature for 24 h, add the TTC staining solution, and determine the MIC values of the antibacterial peptide FK13-a1, geraniol, and eugenol against Escherichia coli, Pseudomonas aeruginosa, and Methicillin-resistant Staphylococcus aureus according to the color change. Design the FIC experiment (based on the CLSI M100 standard) of the two drugs against the pathogenic bacteria according to the MIC values. The specific procedure is to design 8 dilution gradients centered on the single-drug MIC value. The highest concentration is 8 times the single-drug MIC, and the other concentrations decrease in a serial dilution to form a two-dimensional concentration combination.

[0114] The experimental results are as Figure 7 shown. It can be seen that the combined antibacterial effect of geraniol and the antibacterial peptide FK13-a1 against Pseudomonas aeruginosa and Methicillin-resistant Staphylococcus aureus is stronger than that of eugenol.

[0115] Experiment 6 Evaluation of the in vivo pulmonary treatment effect of the membrane-mimicking nanomaterial Tyr-MM@PLGA / G+F

[0116] In sepsis-induced acute lung injury, the lung, as the target organ of infection, is invaded by pathogens, which easily causes severe cell infiltration and lung structure damage. Therefore, in this experiment, the HE staining method is used to observe the lung injury situation to evaluate the treatment effect.

[0117] Experimental animals: SPF-grade male BALB / c (18-20 g) mice, purchased from the Guangdong Provincial Center for Medical Laboratory Animals. The experimental unit's use license is SYXK (Guangdong) 2022-0125), and they are raised in the Medical Laboratory Animal Center of Guangdong Pharmaceutical University (the production license number of experimental animals: SCXK (Guangdong) 2022-0002). Animal experiments strictly abide by the regulations on the management of experimental animals.

[0118] Mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital. After anesthesia, the mice were randomly divided into five groups: Normal group, Model group, PLGA / G+F group, MM@PLGA / G+F group, and Tyr-MM@PLGA / G+F group, with 3 mice in each group. The Model group, PLGA / G+F group, MM@PLGA / G+F group, and Tyr-MM@PLGA / G+F group were all nasally instilled with 50 μl of MRSA (1×10 9 CFU / unit) methicillin-resistant Staphylococcus aureus MRSA (2*10 8 ~5*10 8 CFU / mL), and mice in the Normal group were intravenously injected with approximately 100ul of PBS to establish the model (the drug administration group contained drugs Geraniol: 50mg / kg, FK13-a1: 18.65mg / kg). During this period, the mice's activity decreased, breathing became rapid, and there were wet rales in the lungs, indicating that the model was successful. Four hours later, 100ul of drugs were injected intravenously into the Normal group, Model group, PLGA / G+F group, MM@PLGA / G+F group, and Tyr-MM@PLGA / G+F group for treatment. Mice were sacrificed 24 hours later, and lung tissues were removed from each group for subsequent processing.

[0119] The experimental results are as follows Figure 8 As shown, according to the HE results, the lung tissue damage of the mice in the Model group was severe, but the lung tissue damage of the mice treated with the membrane biomimetic nanomaterial Tyr-MM@PLGA / G+F of the present invention was repaired to a great extent.

[0120] Experiment 7 Evaluation of the antibacterial effect of membrane biomimetic nanomaterial Tyr-MM@PLGA / G+F in vivo

[0121] The treatment of septic infection must first control the number of pathogens in the body to inhibit the occurrence of septic reactions. The lung is the target organ for this experimental modeling. Observing the bacterial content therein will help understand the in vivo antibacterial properties of the material. Therefore, 3 mice were randomly killed 24 hours after infection at a specific time point, and the complete lung tissue was removed under sterile conditions. After weighing, PBS was added in proportion (0.1g lung tissue plus 0.9mL PBS), and a tissue grinder was used to prepare a homogenate to fully release the bacteria. Gradient dilution plating: Take the homogenate and perform a series of dilutions (10 -3 to 10 -7 ) 100 μL of the dilution was spread onto LB agar plates for colony count: After 16-24 hours of incubation, the colony count (CFU) was counted and the bacterial load per milliliter of lung tissue homogenate (CFU / mL) was calculated based on the dilution factor. The number of colonies in the lung homogenate was calculated by colony count to analyze the antibacterial effect of the material.

[0122] The experimental results are as follows Figure 9 As shown, the number of colonies in the lung homogenate of mice in the Model group was large and the infection was more serious. However, after treatment with the membrane biomimetic nanomaterial Tyr-MM@PLGA / G+F of the present invention, the bacterial load in the lungs of mice was significantly reduced.

[0123] Experiment 8 Evaluation of the anti-inflammatory effect of membrane biomimetic nanomaterials in vivo

[0124] The inflammatory cytokine storm is a core element in the pathogenesis of sepsis, occurring throughout the entire course of sepsis. During sepsis, pathogens invade the body, inducing the production of excessive inflammatory factors. These inflammatory factors promote the aggregation of immune cells and their infiltration into lung tissue, activating intracellular signaling pathways and triggering the release of large amounts of cytokines. To evaluate the effects of drugs on inflammatory factors, this study measured the levels of inflammatory factors in bronchoalveolar lavage fluid.

[0125] Mice were divided into groups according to the method of Experiment 6 above: Normal group, Model group, PLGA / G+F group, MM@PLGA / G+F group and Tyr-MM@PLGA / G+F group, with 10 mice in each group. Except for the Normal group, 50 μl MRSA (1×10 9 CFU / mouse). Normal group mice were intravenously injected with approximately 100 μl of PBS for model establishment. During this period, mice exhibited decreased activity, rapid breathing, and pulmonary rales, indicating successful model establishment. After 24 hours of anesthesia, the neck was disinfected and the skin was incised. The muscles were bluntly dissected to expose the trachea. An incision of approximately 2 to 3 mm was made at the distal 2 to 3 cartilage rings of the cricoid cartilage. A puncture needle was then inserted into the trachea, which was then ligated and secured to prevent leakage. Preheated sterile saline (37°C) was drawn into the syringe, with an initial injection of 0.5 to 1 mL. The chest cavity was gently pressed for 10 seconds, then slowly withdrawn. This was repeated three times for a total of approximately 3 mL per mouse.

[0126] The inflammatory factors in the alveolar lavage fluid, such as TNF-α, IL-1β, IL-6, and IL-10, were detected according to the instructions of the ELISA kit (purchased from ELISA Biotechnology Co., Ltd.).

[0127] The experimental results are as follows Figure 10 As shown in the data, compared with the control group, the inflammatory factors TNF-α, IL-1β, and IL-6 in the bronchoalveolar lavage fluid of the PLGA / G+F, MM@PLGA / G+F, and Tyr-MM@PLGA / G+F groups were reduced, with the Tyr-MM@PLGA / G+F group showing the best effect. This indicates that the membrane biomimetic nanomaterial Tyr-MM@PLGA / G+F of the present invention has a good anti-inflammatory effect in vivo.

[0128] Experiment 9 Evaluation of the in vivo immunomodulatory effect of membrane biomimetic nanomaterial Tyr-MM@PLGA / G+F

[0129] The immune response triggered by sepsis has unique characteristics, presenting a cytokine-mediated hyperinflammatory phase followed by an immunosuppressive phase. In the first phase, the immune system is hyperactive, and this overactive immune response is highly likely to cause damage to multiple organs. In the second phase, immune cell dysfunction occurs, and the resulting immunosuppression significantly increases patients' susceptibility to nosocomial infections. To comprehensively evaluate the effects of Tyr-MM@PLGA / G+F on the mouse immune system, this study used flow cytometry to monitor the frequency of immune cell populations in mice treated with MRSA.

[0130] According to the animal modeling method and grouping described in Experiment 6, the mouse lungs were removed 24 hours after administration for flow cytometry analysis (Sony ID7000), and the detection indicators included neutrophils, regulatory T cells, and CD4+ and CD8+.

[0131] The specific steps include: Mice were anesthetized with pentobarbital and then sacrificed by cervical dislocation. The lungs were removed intact and quickly immersed in clean PBS. Using a scalpel, the lung tissue was minced into small pieces approximately 0.2 cm² in size. The pieces were then digested with type I collagenase at 37°C for 30 minutes. Upon completion of digestion, the lung tissue was transferred to a 200-mesh sieve and gently ground with a tissue grinder until no visible red lumps remained on the sieve. The sieve was then rinsed with 15 mL of PBS and the resulting fluid was collected into a 15 mL centrifuge tube. The tube was centrifuged at 300 g for 5 minutes, and the supernatant was discarded. 2 mL of 1x red blood cell lysis buffer was added to the tube to resuspend the cells. The lysis reaction was allowed to proceed at room temperature for 2 to 3 minutes. After the reaction was complete, 10 mL of PBS was immediately added and the tube was centrifuged again at 300 g for 5 minutes. The supernatant was discarded. Resuspend the lung cells in cell staining buffer and filter the resulting cell suspension through a 200-mesh sieve again. After filtration, count the cells and adjust the cell concentration to 1 × 10 7 Finally, cell analysis was performed using a Sony ID700 flow cytometer and accompanying software according to the kit instructions. Data analysis was performed in Flow Cytometry v10.8.1.

[0132] The experimental results are as follows Figure 11As shown, compared with the control group, the PLGA / G+F group, the MM@PLGA / G+F group, and the Tyr-MM@PLGA / G+F group significantly restored the number of CD4+ and CD8+ cells and regulatory T cells, while reducing the level of neutrophils. Among them, the Tyr-MM@PLGA / G+F group had the best effect. This showed a good immunomodulatory effect, indicating that the membrane biomimetic nanomaterial Tyr-MM@PLGA / G+F provided by the present invention significantly reduced the occurrence of inflammatory reactions during the inflammatory cytokine storm.

[0133] In summary, the Tyr-MM@PLGA / G+F membrane biomimetic nanomaterial provided by the present invention has both septic lung injury treatment and inflammatory responsiveness functions. It can respond to inflammatory signals at the lesion site of septic lung injury, target the lesion site, and bring the antimicrobial peptide FK13-a1 and Geraniol into the lesion site for release. At the same time, under inflammatory conditions, the material degrades, exposing the active ingredients so that the antimicrobial peptide FK13-a1 and geraniol act synergistically, thereby correspondingly alleviating and treating various conditions such as inflammation, oxidative stress, and endothelial damage caused by septic lung injury.

[0134] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Anyone skilled in the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by one of ordinary skill in the art without departing from the spirit and technical principles disclosed herein are intended to be covered by the claims of the present invention.

Claims

1. A method for preparing a membrane biomimetic nanomaterial for treating septic lung injury, characterized in that: The steps include: S1. Preparation of Targeting Peptide DSPE-PEG-Tyr Storage Solution: The targeting peptide DSPE-PEG-Tyr powder was added to a certain amount of PMSF to prepare a 0.1-10 mg / mL targeting peptide DSPE-PEG-Tyr storage solution for later use; Preparation of S2, MM-Tyr: The target peptide DSPE-PEG-Tyr stock solution prepared in step S1 was added to a 0.5-2 mg / mL PBS solution of macrophage membrane MM and incubated at 4°C for 12 hours to finally prepare MM-Tyr; S3. Preparation of solution: Dissolve geraniol in chloroform to prepare a geraniol solution with a final concentration of 5-10 μg / mL, and set aside; dissolve the antimicrobial peptide FK13-a1 in water to prepare an antimicrobial peptide FK13-a1 aqueous solution with a final concentration of 12-16 μg / mL, and set aside; prepare a PVA-CS storage solution containing 0.8-1.2% polyvinyl alcohol (PVA) and 0.1-0.3% chitosan (CS), and set aside; S4. Loading of active ingredients: The antimicrobial peptide FK13-a1 aqueous solution prepared in step S3 is added to the geraniol solution and subjected to a first sonication to prepare an O / W nanoemulsion; the O / W nanoemulsion is then added to the PVA-CS storage solution prepared in step S3 and subjected to a second sonication to prepare a nanoemulsion PLGA / G+F; S5. Construction of membrane biomimetic nanomaterial Tyr-MM@PLGA / G+F: The MM-Tyr prepared in step S2 and the nanoemulsion PLGA / G+F prepared in step S3 were gently mixed at a mass ratio of 1:(0.2-0.8) and incubated overnight at 2-6°C; the solution was extruded through a PC membrane with a pore size of 200 nm and collected to prepare a membrane biomimetic nanomaterial Tyr-MM@PLGA / G+F.

2. The preparation method according to claim 1, characterized in that The targeting peptide DSPE-PEG-Tyr powder in step S1 is a targeting peptide DSPE-PEG-Tyr powder with a purity greater than 95% HPLC purity.

3. The preparation method according to claim 1, characterized in that The cell membranes derived from mouse mononuclear macrophage leukemia cells in step S2 are obtained by culturing Raw264.7 cells, collecting, and extracting the cell membranes of Raw264.7 cells.

4. The preparation method according to claim 3, characterized in that The culturing of Raw264.7 cells comprises: culturing the Raw264.7 cells in a 35-45° C., 2-8% CO2 environment using a complete DMEM culture medium; when the cell density reaches about 75-80%, discarding the original culture medium; rinsing the cells 2-3 times with a PBS buffer solution to completely remove the cells; collecting the cell suspension; and temporarily storing the cell suspension in a -80-100° C. low-temperature refrigerator for future use.

5. The preparation method according to claim 1, characterized in that The amino acid sequence of the antimicrobial peptide FK13-a1 in step S3 is shown in SEQ.ID.NO.

1.

6. The preparation method according to claim 1, characterized in that The conditions of the first ultrasound in step S4 are 2 to 4 seconds each time, 1 to 3 seconds interval, and an amplitude of 20 to 40%.

7. The preparation method according to claim 1, characterized in that The conditions for the second ultrasound in step S4 are 2 to 4 seconds each time, 1 to 3 seconds interval, and an amplitude of 20 to 40%.

8. A membrane biomimetic nanomaterial prepared by the preparation method according to any one of claims 1 to 7.

9. An application of the membrane biomimetic nanomaterial according to claim 8, characterized in that: The membrane bionic nanomaterial is used for preparing medicine for treating septic lung injury.

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