Cellular-imitated nano preparation for treating acute respiratory distress syndrome

Abstract

The invention discloses an imitated intercorial nano preparation for treating acute respiratory distress syndrome, and belongs to the technical field of biological medicine. The internalized nano preparation comprises an active component, a liposome phospholipid bilayer and a target head, wherein the active component consists of an ROS (reactive oxygen species) removing drug and an anti-inflammatory drug. According to the invention, efficient and accurate targeting of the nano-preparation is realized by innovatively applying an imitated interment effect, an apoptotic cell marker is modified on the surface of the liposome, and an'eat me 'signal is released to simulate a natural biological process that macrophages clear apoptotic cells to be recognized and swallowed by alveolar macrophages, so that efficient and specific targeting is realized, and the targeting effect of the nano-preparation is improved. Meanwhile, up-regulation of PS receptor expression of macrophages at the inflammation part further enhances the targeting effect, and a new way is provided for efficient delivery of acute respiratory distress syndrome medicine preparations and remodeling of pathological microenvironment.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a cell-mimicking nano preparation for treating acute respiratory distress syndrome. Background Art

[0002] Acute respiratory distress syndrome (ARDS) is an acute inflammatory lung disease caused by a variety of etiologies, including sepsis, lung infection, and septicemia. In severe cases, respiratory failure may occur. Pathological manifestations primarily include damage to the alveolar endothelium and epithelial barrier, mediated by innate immune cells, and the accumulation of proteinaceous edema fluid within the interstitium and alveoli. Under stressful conditions, alveolar immune cells secrete proinflammatory mediators and reactive oxygen species, creating a vicious cycle that further exacerbates the inflammatory microenvironment of ARDS. Due to the complex pathological microenvironment, there are currently no effective medications for ARDS. Clinical treatment is primarily supportive, including mechanical ventilation, vasodilators, and potent anti-inflammatory drugs.

[0003] Currently, commonly used clinical treatments can only temporarily control the progression of the disease and alleviate pain. The use of mechanical ventilation can cause mechanical damage to the patient's lungs. Commonly used anti-inflammatory drugs, such as glucocorticoids, have significant systemic side effects and can easily cause immunosuppression. Therefore, there is an urgent need to design a drug delivery system that can efficiently deliver drugs, disrupt the harmful crosstalk between ROS and inflammatory cells in the ARDS pathological microenvironment, and effectively repair the lung's normal immune barrier. Summary of the Invention

[0004] One of the purposes of the present invention is to provide a cell burial mimicking nanoformulation, comprising an active ingredient, a liposome phospholipid bilayer and a target head; the active ingredient is composed of a ROS scavenging drug and an anti-inflammatory drug, the liposome phospholipid bilayer is composed of cholesterol and lecithin, and the target head is selected from any one or more of phosphatidylserine, hyaluronic acid, folic acid, mannose or galactose.

[0005] Furthermore, the ROS scavenging drug is selected from any one or more of nanozymes, superoxide dismutase analogs, vitamin E, vitamin C, idebenone, glutathione or glutathione precursors.

[0006] Furthermore, the ROS scavenging drug is a nanozyme, which is made of albumin and a strong oxidizing agent.

[0007] Furthermore, the albumin is selected from any one or more of bovine serum albumin, human serum albumin, mouse serum albumin or ovalbumin.

[0008] Furthermore, the strong oxidizing agent is selected from any one or more of potassium permanganate (KMnO4), potassium dichromate (K2CrO7), potassium periodate (KIO4), manganese dioxide (MnO2) and peroxy acid.

[0009] Furthermore, the anti-inflammatory drug is selected from any one or more of tanshinone IIA, baicalin, berberine, andrographolide, curcumin, matrine, chlorogenic acid or triptolide.

[0010] Furthermore, the lecithin is selected from any one or more of soybean lecithin, egg yolk lecithin, hydrogenated soybean lecithin (HSPC), distearoylphosphatidylcholine (DSPC) or dipalmitoylphosphatidylcholine (DPPC).

[0011] Furthermore, the mass ratio of the phospholipid to cholesterol is 5:1 to 1:2, the mass ratio of the target head to lecithin is 10:1 to 1:1, the ratio of the mass of the anti-inflammatory drug to the sum of the masses of cholesterol, lecithin and phosphatidylserine is 1:5 to 1:40, and the mass ratio of the ROS scavenging drug to the anti-inflammatory drug is 10:1 to 1:1.

[0012] In a specific embodiment of the present invention, the ROS scavenging drug is made of BSA and KMnO4, the anti-inflammatory drug is tanshinone IIA, and the target head is phosphatidylserine (PS). Furthermore, the mass ratio of phospholipid to cholesterol is 5:1 to 1:2, the mass ratio of phosphatidylserine to phosphatidylcholine is 10:1 to 1:1, the ratio of the mass of tanshinone IIA to the sum of the masses of cholesterol, phosphatidylcholine, and phosphatidylserine is 1:5 to 1:40, and the mass ratio of the ROS scavenging drug to the anti-inflammatory drug is 10:1 to 1:1.

[0013] In the present invention, the preparation method of the liposome phospholipid bilayer can be prepared by a common method in the art without limitation, such as thin film dispersion method, reverse evaporation method, extrusion method, high pressure homogenization method or organic solvent injection method.

[0014] In a specific embodiment of the present invention, the preparation method of the cell-mimicking nanoformulation comprises the following steps:

[0015] Step 1, preparation of ROS scavenger nanoparticles: slowly add KMnO4 aqueous solution dropwise to the stirred BSA aqueous solution under ice bath conditions, then remove the ice bath after the solution addition is complete, and continue stirring at room temperature for 2 h to form nanoparticles;

[0016] Step 2, preparation of nanoformulation: dissolving phosphatidylserine, lecithin, cholesterol and tanshinone IIA in an organic solvent, rotary evaporating under reduced pressure to form a thin film, adding an aqueous solution containing ROS scavenger nanoparticles to hydrate the thin film, and ultrasonically homogenizing to obtain a liposome solution with good uniformity.

[0017] Specifically, the concentration of the ROS scavenger nanoparticle solution is controlled to be 1-10 mg / mL. The concentration of the organic solution of tanshinone IIA is controlled to be 0.1-1 mg / mL. The organic solvent for dissolving the lipid and tanshinone IIA is selected from any one or more of chloroform, dichloromethane, methanol, ethanol, ether, acetone, dimethyl ethyl sulfone, and N,N-dimethylformamide.

[0018] The second purpose of the present invention is to provide the use of the above-mentioned cell-mimicking nanoformulation in the preparation of drugs for the prevention and treatment of acute respiratory distress syndrome.

[0019] The design principles of the present invention are as follows:

[0020] Because the inflamed areas of the lungs are rich in inflammatory macrophages, the present invention selects phosphatidylserine (PS), a marker of apoptotic cells, as a target, leveraging a natural biological process to be specifically recognized by macrophages to help target the inflamed areas. Secondly, due to the complex immune microenvironment in the lungs of patients with acute respiratory distress syndrome, ROS, in addition to being a product of inflammation, also activates the NF-κB and NLRP3 pathways to promote the release of more inflammatory factors, forming a vicious cycle. Therefore, it is necessary to use both anti-inflammatory drugs and ROS scavenging drugs to interrupt the vicious cycle. Tanshinone IIA is a fat-soluble diterpene quinone compound extracted from the traditional Chinese medicine Salvia miltiorrhiza. As the main active ingredient of Salvia miltiorrhiza, it is widely used in cardiovascular protection, anti-inflammatory, antioxidant and other fields. It can block the activation of NF-κB, inhibit the activation of inflammasome NLRP3, and effectively inhibit the release of inflammatory factors. A nanozyme BSA-MnO2 with excellent dispersibility and high biocompatibility was prepared by a one-step method. BSA-MnO2 was encapsulated in the aqueous phase of liposomes, and Tanshinone IIA was loaded into the phospholipid bilayer of liposomes to achieve co-encapsulation of fat-soluble drugs and water-soluble drugs in the same delivery system.

[0021] Compared with the prior art, the present invention has the following significant advantages:

[0022] (1) The present invention innovatively uses the mimetic cell burial effect to achieve efficient and precise targeting of nanoformulations. The apoptotic cell marker phosphatidylserine (PS) is modified on the surface of liposomes. By releasing the "eat me" signal, the natural biological process of macrophages clearing apoptotic cells is simulated and recognized and phagocytosed by alveolar macrophages, achieving efficient and specific targeting. At the same time, the upregulation of PS receptor expression in macrophages at the inflammatory site further enhances the targeting effect, providing a new approach for the efficient delivery of drug preparations for acute respiratory distress syndrome and the reshaping of the pathological microenvironment.

[0023] (2) The present invention simultaneously inhibits the release of pro-inflammatory cytokines and clears ROS, breaks the vicious cycle between inflammatory cytokines and ROS, polarizes pro-inflammatory macrophages into anti-inflammatory macrophages, thereby effectively reshaping the malignant microenvironment of ARDS and repairing lung function. It has important application prospects for ARDS secondary to severe novel coronavirus infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Figures show the appearance, particle size, potential, and stability of the nanoformulation in Example 1. A is a transmission electron microscopy image of BMTPLs; B is a particle size distribution diagram of BSA, BM, and BMTPLs; C is a zeta potential diagram of BSA, BM, and BMTPLs; and D is a graph showing the particle size and PDI stability of BMTPLs in saline for 7 days.

[0025] Figure 2 This is an analysis chart of the ROS scavenging ability of the nanoformulation BM in Example 1.

[0026] Figure 3 FITC fluorescence quantitative graphs of nanoformulations with and without PS modification.

[0027] Figure 4 Cell uptake diagrams of nanoformulations with and without PS modification at different time points.

[0028] Figure 5 This is the survival status of RAW264.7 cells after co-incubation with the nanoformulation BMTPLs in the example.

[0029] Figure 6 This is a fluorescence confocal image of intracellular DCFH-DA after inflammatory cells were co-incubated with the nanoformulation in Example 1.

[0030] Figure 7 This is a graph showing the detection results of MDA, GSH, SOD and CAT contents in the cell culture supernatant.

[0031] Figure 8 Quantitative results of the expression of inflammatory factors IL-18, IL-1β, IL-6 and TNF-α in cells.

[0032] Figure 9 The figure shows the protein quantification of NLRP3, NF-κB and IL-1β in cells.

[0033] Figure 10 Quantitative graph of p-NF-κB protein in the activated state of cells.

[0034] Figure 11 Immunofluorescence images of CD86 and CD206.

[0035] Figure 12 To construct pathological sections and immunofluorescence images of the lungs after treatment with different nanoformulations in mice with acute respiratory distress syndrome.

[0036] Figure 13 This is a graph showing respiratory function testing in mice with acute respiratory distress syndrome after treatment with different nanoformulations. DETAILED DESCRIPTION

[0037] The preferred embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that the following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0038] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0039] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0040] Example 1

[0041] Preparation of nanoparticles

[0042] 1. The preparation method of BSA-MnO2 is as follows:

[0043] In an ice bath, 25 mL of a 4 mg / mL KMnO₄ solution was evenly and slowly added to 25 mL of a 20 mg / mL BSA solution. After the addition was complete, the ice bath was removed and the mixture was stirred at room temperature for 2 hours until the solution color changed from wine red to brown-black. Finally, the resulting nanoparticles were purified using a dialysis bag (MWCO = 14,000 Da) with ultrapure water at room temperature for 48 hours to remove unreacted small molecules. After dialysis, the mixture was centrifuged at 14,000 rpm / min for 20 minutes to remove possible aggregates. The final ROS-scavenging nanoparticles, BSA-MnO₂ (BM), were obtained and freeze-dried and stored at 4°C until further use.

[0044] 2. The preparation method of BSA-MnO2@Tan-IIA / PLs is as follows:

[0045] 5.2 mg of phosphatidylserine (PS), 2.195 mg of soy lecithin, 2.61 mg of cholesterol, and 1 mg of tanshinone IIA were dissolved in 2 mL of chloroform / methanol solution (volume ratio 9:1) and transferred to a 50 mL eggplant-shaped flask. The organic solvent was evaporated under reduced pressure using a rotary evaporator at 37°C until a uniform, transparent film formed on the inner wall of the flask. Subsequently, 2 mL of a 2 mg / mL BSA-MnO2 aqueous solution was added and hydrated at 45°C for 15 min. The resulting solution was homogenized using an ultrasonic cell disruptor for 5 min. This yielded the nanoformulation BSA-MnO2@Tan-IIA / PLs (BMTPLs).

[0046] like Figure 1 As shown in A and B, the transmission electron microscopy results show that the morphology of BSA-MnO2@Tan-IIA / PLs is a very small and uniform sphere, and the dynamic light scattering particle size is 123.44±2.67. Figure 1 Figure C shows that BSA-MnO2@Tan-IIA / PLs can exist stably in normal saline for 7 days, which means that it has good stability under physiological conditions. Figure 1 B in the figure is the potential of several preparations during the synthesis of BSA-MnO2@Tan-IIA / PLs, all between -30mV and 0mV.

[0047] 3. The preparation method of Tan-IIA / PLs is as follows:

[0048] Using only Tanshinone IIA as the drug ingredient, 5.2 mg of phosphatidylserine, 2.195 mg of soy lecithin, 2.61 mg of cholesterol, and 1 mg of Tanshinone IIA were dissolved in 2 mL of chloroform / methanol solution (volume ratio 9:1). The mixture was transferred to a 50 mL eggplant-shaped flask. The organic solvent was evaporated under reduced pressure using a rotary evaporator at 37°C until a uniform, transparent film formed on the inner wall of the flask. Subsequently, 2 mL of ultrapure water was added and the mixture was hydrated at 45°C for 15 minutes. The resulting solution was homogenized using an ultrasonic cell disruptor for 5 minutes. This yielded the nanoformulation Tan-IIA / PLs (TPLs).

[0049] 4. The preparation method of BSA-MnO2 / PLs is as follows:

[0050] Using only BSA-MnO2 as the drug component, 5.2 mg of phosphatidylserine, 2.195 mg of soy lecithin, and 2.61 mg of cholesterol were weighed and dissolved in 2 mL of chloroform / methanol solution (volume ratio of 9:1). The mixture was transferred to a 50 mL eggplant-shaped flask. The organic solvent was evaporated under reduced pressure on a rotary evaporator at 37°C until a uniform, transparent film formed on the inner wall of the flask. Subsequently, 2 mL of an aqueous solution containing BSA-MnO2 was added and hydrated at 45°C for 15 minutes. The resulting solution was homogenized using an ultrasonic cell disruptor for 5 minutes. This yielded the nanoformulation BSA-MnO2 / PLs (BMPLs).

[0051] 5. The preparation method of BSA-MnO2@Tan-IIA / Ls is as follows:

[0052] 2.195 mg of soybean lecithin, 2.61 mg of cholesterol, and 0.48 mg of Tanshinone IIA were dissolved in 2 mL of chloroform / methanol solution (volume ratio 9:1) and transferred to a 50 mL eggplant-shaped flask. The organic solvent was evaporated under reduced pressure on a rotary evaporator at 37°C until a uniform, transparent film formed on the inner wall of the flask. Subsequently, 2 mL of a 2 mg / mL BSA-MnO2 aqueous solution was added and hydrated at 45°C for 15 minutes. The resulting solution was homogenized using an ultrasonic cell disruptor for 5 minutes. This yielded the nanoformulation BSA-MnO2@Tan-IIA / Ls (BMTLs).

[0053] 6. The preparation methods of BSA-MnO2@Tan-IIA / FITC / Ls and BSA-MnO2@Tan-IIA / FITC / PLs are as follows:

[0054] 2.195 mg of soybean lecithin, 2.61 mg of cholesterol, 0.48 mg of FTTC, and 0.48 mg of tanshinone IIA were weighed and dissolved in 2 mL of chloroform / methanol solution (volume ratio of 9:1), and then transferred to a 50 mL eggplant-shaped flask. The rest of the process was the same as in Example 1.2 to obtain the nanoformulation BSA-MnO2@Tan-IIA / FITC / Ls (BMTLs-FITC).

[0055] 5.2 mg of phosphatidylserine, 2.195 mg of soy lecithin, 2.61 mg of cholesterol, 1 mg of FITC, and 1 mg of tanshinone IIA were weighed and dissolved in 2 mL of chloroform / methanol solution (volume ratio of 9:1), and then transferred to a 50 mL eggplant-shaped flask. The rest of the process was the same as that in Example 1.2 to obtain the nanoformulation BSA-MnO2@Tan-IIA / FITC / PLs (BMTPLs-FITC).

[0056] Example 2

[0057] Investigation of ROS scavenging ability of BSA-MnO2 nanoparticles

[0058] BSA-MnO2 (hereafter abbreviated as BM) was diluted with ultrapure water to various concentrations (0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 mg / mL) to prepare a 10 mM H2O2 solution. The BM-related H2O2 scavenging ability was then assessed using a hydrogen peroxide detection kit. ABTS working solutions were prepared by mixing a 1 / 1 volume ratio of ABTS stock solution (7 mM) with a 2.45 mM K2S2O3 solution at room temperature in the dark for 16 hours. The ABTS working solutions were then incubated with various BM concentrations for 5 minutes, followed by measuring absorbance at 734 nm. The total antioxidant capacity of BM at various concentrations was determined.

[0059] like Figure 2 The results showed that the H2O2 clearance rate and total antioxidant capacity were proportional to the BM concentration. When the concentration was 0.5 mg / mL, the H2O2 clearance rate was close to 100%.

[0060] Example 3

[0061] Characterization of surface modification of nanoformulations

[0062] In the early stages of apoptosis, phosphatidylserine flips from the inside to the outside of the cell membrane. Externalized phosphatidylserine, the most universal marker for phagocytes to identify apoptotic cells, can be rapidly recognized and cleared by phagocytes. Annexin V has a high affinity for phosphatidylserine and is commonly used to detect early apoptotic cells. Therefore, Annexin V-FITC was incubated with PS-modified BMTPLs and non-PS-modified BMTLs for 30 minutes, then transferred to an ultrafiltration tube and centrifuged at 3000 rpm / min for 10 minutes to collect the preparation. The preparation was diluted to the same volume with pure water and then added to a 96-well black microplate and the fluorescence intensity was measured using a microplate reader. Figure 3 The fluorescence intensity quantitative graph of the two preparations after incubation with Annexin V-FITC shows that there is a large amount of phosphatidylserine on the surface of BMTPLs.

[0063] Example 4

[0064] Investigation of the targeting properties of nanoformulations

[0065] To evaluate the cellular uptake effect of phosphatidylserine-modified nanoformulations, RAW264.7 cells were seeded in glass-bottomed dishes overnight, and a 500 ng / mL lipopolysaccharide solution was prepared in serum-free 1640 medium. The cells were stimulated to polarize into pro-inflammatory M1 macrophages, and then co-incubated with FITC-labeled BMTLs and BMTPLs for 2 h, 4 h, and 8 h, respectively. The medium was discarded, and the cells were washed twice with 1 mL of PBS. The cells were fixed with 1 mL of 4% paraformaldehyde for 15 min, then washed twice with PBS, and the cell nuclei were stained with DAPI. The fluorescence intensity was observed by laser confocal microscopy and photographed. Figure 4 The results showed that the uptake of phosphatidylserine-modified BMTPLs nanoparticles was significantly higher than that of BMTLs, and the cellular uptake gradually increased with time.

[0066] Example 5

[0067] Investigation of the biosafety of nanoparticles

[0068] RAW264.7 cells were seeded in six-well plates overnight, the culture medium was discarded, and a 500 ng / mL lipopolysaccharide solution was prepared in serum-free 1640 culture medium. The lipopolysaccharide solution was administered separately to stimulate the cells to polarize into pro-inflammatory M1 macrophages. Different concentrations of BMTPLs solutions (12.5, 25, 50, 100 μg / mL) were prepared in 1640 culture medium containing 10% FBS. After incubation with the cells for 24 hours, Calcein AM and PI were used to perform dual fluorescence staining of live and dead cells. The intensity of the two fluorescences was observed under an inverted fluorescence microscope, and the cell activity and cytotoxicity were detected based on the fluorescence intensity. Figure 5 The results showed that BMTPLs were highly safe for cells.

[0069] Example 6

[0070] Analysis of the intracellular antioxidant effect of nanoparticles

[0071] DCFH-DA is a fluorescent probe for detecting reactive oxygen species. It freely penetrates the cell membrane and, upon entry, is hydrolyzed by intracellular esterases to produce DCFH, which is subsequently oxidized by intracellular ROS to produce fluorescent DCF. The fluorescence intensity of DCF is used to quantify the intracellular ROS content. RAW 264.7 cells were evenly plated in glass-bottomed dishes and six-well plates and placed in an incubator overnight to allow complete cell attachment. The cells were divided into the PBS group, the Tan-IIA / PLs group (hereinafter abbreviated as TPLs), the BSA-MnO2 / PLs group (hereinafter abbreviated as BMPLs), and the BSA-MnO2@Tan-IIA / PLs group (hereinafter abbreviated as BMTPLs). The culture medium was discarded, and a 500 ng / mL lipopolysaccharide solution was prepared in serum-free 1640 medium. This solution was administered to stimulate the cells to polarize into pro-inflammatory M1 macrophages. After 6 hours, the solution was discarded, and different concentrations of the nanoformulations prepared in Example 1, TPLs, BMPLs, and BMTPLs, were prepared in complete culture medium. 1 mL of each solution was added to the corresponding dish or well for incubation. DCFH-DA staining was then performed for 30 minutes, and fluorescence intensity was observed using a laser confocal microscope and photographed.

[0072] like Figure 6 As shown, after inflammation was induced in the cells, the intracellular DCF fluorescence intensity of the cells administered with the preparation BMTPLs was significantly reduced.

[0073] Malondialdehyde (MDA) is one of the main products of lipid peroxidation, mainly produced by ROS oxidizing unsaturated fatty acids in cell membranes or organelle membranes. Usually, high MDA levels indicate that cell membrane lipids have been significantly oxidatively damaged. Reduced glutathione (GSH) is an important non-enzymatic antioxidant in cells that can directly remove ROS. Usually, low GSH levels indicate that the cell's antioxidant capacity is reduced and it is in a state of oxidative stress. Superoxide dismutase (SOD) is a type of enzyme that can remove O2 -·Antioxidant enzyme, catalase (CAT) is a key enzyme for removing hydrogen peroxide. RAW264.7 was selected as the research object, and the cells were evenly plated in a six-well plate and placed in an incubator overnight to allow the cells to completely adhere to the wall. The groups were set as PBS group, TPLs group, BMPLs group and BMTPLs group respectively. The culture medium was discarded, and a lipopolysaccharide solution with a concentration of 500ng / mL was prepared with serum-free 1640 culture medium. The drugs were administered separately to stimulate the polarization of the cells into pro-inflammatory M1 macrophages. After 6h, the drug solution was discarded, and the drug solution of the nanoformulation TPLs, BMPLs and BMTPLs prepared in Example 1 with different concentrations was respectively configured using complete culture medium, and 1mL was added to the corresponding wells for incubation. After 24h, the cell culture medium was collected, and the suspended cells in the culture medium were removed by centrifugation at 2000rpm / min for 5min. The cell culture supernatant was collected and measured with MDA, GSH, SOD and CAT detection kits respectively.

[0074] Figure 7 The results of the analysis of MDA, GSH, SOD, and CAT in the cell culture supernatant show that after LPS-induced inflammatory oxidative stress in RAW264.7 cells, MDA levels decreased, cell membranes suffered oxidative damage, GSH, SOD, and CAT levels decreased, and the cells' antioxidant capacity significantly decreased. BMTPLs can effectively reduce cellular oxidative stress and restore the cells' own antioxidant capacity.

[0075] Example 7

[0076] Analysis of the anti-inflammatory effect of nanoformulations in vitro

[0077] 1. RAW264.7 cells were seeded in a six-well plate overnight and divided into PBS group, TPLs group, BMPLs group and BMTPLs group. 500 ng / mL lipopolysaccharide solution was used to induce cell polarization to M1 type. After 6 hours, 1 mL of the nanoformulation TPLs, BMPLs and BMTPLs prepared in Example 1 were added to the corresponding wells for incubation. After 24 hours, the cells in different groups were collected and the cell RNA was extracted according to the standard procedure in the instruction manual (KeyGen Biotechnology, KGF5101-100) and then reverse transcribed into cDNA. Finally, it was mixed with different primers for qPCR to quantify the expression of inflammatory factors IL-18, IL-1β, IL-6 and TNF-α in the cells.

[0078] 2. Cells from different groups were collected according to the above procedure and lysed on ice in lysis buffer for 30 minutes. Protein was extracted by vortexing every 4 minutes. Protein was quantified using the BCA assay. Protein components were quantitatively analyzed by Western blot, including the internal reference protein GAPDH, the inflammasome protein NLRP3, the important inflammatory pathway protein NF-κB, the inflammatory cytokine protein IL-1β, and the activated inflammatory pathway protein p-NF-κB.

[0079] like Figure 8 As shown in Figure 3, the expression of inflammatory factors IL-18, IL-1β, IL-6, and TNF-α in cells was significantly reduced after treatment with the BMTPLs formulation. Figure 9 and Figure 10 It was shown that the expression of inflammation-related proteins was significantly reduced after BMTPLs treatment.

[0080] Example 8

[0081] Analysis of the macrophage reprogramming effect of nanoformulations

[0082] RAW264.7 cells were seeded in glass-bottomed dishes overnight and divided into PBS, TPLs, BMPLs, and BMTPLs groups. The culture medium was discarded, and a 500 ng / mL lipopolysaccharide solution was prepared in serum-free 1640 medium. The cells were polarized into pro-inflammatory M1 macrophages. After 6 hours, the solution was discarded, and the nanoformulations TPLs, BMPLs, and BMTPLs prepared in Example 1 were prepared in complete culture medium. 1 mL of each solution was added to the corresponding wells for incubation. 24 hours after administration, the culture medium was discarded, and the cells were washed twice with 1 mL of PBS. The cells were fixed with 1 mL of 4% paraformaldehyde for 15 minutes and then permeabilized with 0.1% Triton X-100. After washing with PBS three times, cells were blocked with 1% BSA at room temperature for 30 minutes, then incubated with antibodies PE-CD86 and APC-CD206 at 4°C overnight, then washed twice with PBS and stained with DAPI for nuclei. Finally, the fluorescence intensity of PE and APC was observed by laser confocal microscopy and photographed. Figure 11 As shown, the expression of CD206 increased in the BMTPLs group, and the cells polarized from M1 to M2.

[0083] Example 9

[0084] Analysis of the effect of nanoformulation on improving lung inflammation in mice with acute respiratory distress syndrome

[0085] Balb / c mice were intubated to develop acute respiratory distress syndrome (ARDS). LPS was administered to the mice's lungs at a concentration of 7.5 mg / kg via endotracheal intubation. Six hours after modeling, the mice were randomly assigned to four groups: PBS, TPLs, BMPLs, and BMTPLs. A negative control, the sham group, was also established, along with normal mice that had not undergone modeling. Correspondingly, different nanoformulations were administered via tracheal instillation. Twenty-four hours after treatment, the mice were euthanized, and lung tissue was dissected and analyzed by H&E staining and immunofluorescence. Figure 12 Figure A is a pathological section of the mouse lungs. The results showed that the mice had severe lung damage after modeling, mainly manifested by thickening of the alveolar septum, increased hyaline membranes, and neutrophil aggregation. After administration of the drugs TPLs and BMPLs, the lung damage was alleviated, but the BMTPLs group showed a more significant therapeutic effect. Figure 12 Figure B shows immunofluorescence staining of mouse lung tissue. The results show that compared with the other groups, the BMTPLs group had lower iNOS expression and higher CD206 protein expression, indicating that BMTPLs effectively improved the lung inflammatory microenvironment. The nanoformulation can effectively treat acute respiratory distress syndrome.

[0086] Example 10

[0087] Analysis of the effect of nanoformulation on restoring respiratory function in mice with acute respiratory distress syndrome

[0088] Balb / c mice were intubated to induce acute respiratory distress syndrome (ARDS) and administered lipopolysaccharide (LPS) at a concentration of 7.5 mg / kg. Six hours after intubation, the mice were randomly assigned to four groups: PBS, TPLs, BMPLs, and BMTPLs. Corresponding nanoformulations were administered via tracheal instillation. After 24 hours of treatment, respiratory function in the treated mice was assessed using whole-body plethysmography (WBP). Figure 13 The results showed that compared with the PBS group, the minute ventilation (MV), tidal volume (TV) and relaxation time (tR) of the TPLs group, BMPLs group and BMTPLs group were significantly restored. Among them, the final preparation BMTPLs group had the best effect, indicating that the nanoformulation can effectively restore the respiratory function of mice with acute respiratory distress syndrome.

Claims

1. A cell-mimicking nanoparticle preparation, characterized in that: The invention comprises an active ingredient, a liposome phospholipid bilayer and a target head; the active ingredient is composed of a ROS scavenging drug and an anti-inflammatory drug, the liposome phospholipid bilayer is composed of cholesterol and lecithin, and the target head is selected from any one or more of phosphatidylserine, hyaluronic acid, folic acid, mannose or galactose.

2. The cell-mimicking nano preparation according to claim 1, characterized in that: The ROS scavenging drug is selected from any one or more of nanozymes, superoxide dismutase analogs, vitamin E, vitamin C, idebenone, glutathione or glutathione precursors.

3. The cell-mimicking nano preparation according to claim 2, characterized in that: The ROS scavenging drug is a nanozyme, which is made of albumin and a strong oxidizing agent.

4. The cell-mimicking nano preparation according to claim 3, characterized in that The albumin is selected from any one or more of bovine serum albumin, human serum albumin, mouse serum albumin or ovalbumin.

5. The cell-mimicking nano preparation according to claim 3, characterized in that: The strong oxidizing agent is selected from any one or more of potassium permanganate, potassium dichromate, potassium periodate, manganese dioxide or peroxy acid.

6. The cell-mimicking nano preparation according to claim 1, characterized in that: The anti-inflammatory drug is selected from any one or more of tanshinone IIA, baicalin, berberine, andrographolide, curcumin, matrine, chlorogenic acid or triptolide.

7. The cell-mimicking nano preparation according to claim 1, characterized in that: The lecithin is selected from any one or more of soybean lecithin, egg yolk lecithin, hydrogenated soybean lecithin, distearoylphosphatidylcholine or dipalmitoylphosphatidylcholine.

8. The cell-mimicking nano preparation according to claim 1, characterized in that: The mass ratio of the phospholipid to cholesterol is 5:1-1:2, the mass ratio of the target head to lecithin is 10:1-1:1, the ratio of the mass of the anti-inflammatory drug to the sum of the masses of cholesterol, lecithin and phosphatidylserine is 1:5-1:40, and the mass ratio of the ROS scavenging drug to the anti-inflammatory drug is 10:1-1:

1.

9. The method for preparing the cell-mimicking nanoformulation according to any one of claims 1 to 8, characterized in that: The following steps are involved: The target head, lecithin, cholesterol and anti-inflammatory drugs are dissolved in an organic solvent, and rotary evaporated under reduced pressure to form a film. An aqueous solution containing ROS scavenging drugs is added to hydrate the film, and the cell burial-mimicking nanoformulation is obtained after ultrasonic homogenization.

10. Use of the cell-burying-mimicking nanoformulation according to any one of claims 1 to 8 in the preparation of drugs for the prevention and treatment of acute respiratory distress syndrome.

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