Preparation method of macrophage membrane modified MPDA-CeO2 bionic nano-composite and application of MPDA-CeO2 bionic nano-composite in treatment of acute lung injury

Abstract

The invention provides a preparation method of a macrophage membrane modified MPDA-CeO2 bionic nano-composite and application of the MPDA-CeO2 bionic nano-composite in treatment of acute lung injury. The nano-composite is a novel nano-material designed by applying a bionics principle; the surface of the mesoporous polydopamine-loaded cerium dioxide nano-composite is successfully coated with the extracted macrophage membrane micro-capsule, and the excellent antioxidant functions of the mesoporous polydopamine and the cerium dioxide are combined to realize active targeting on the lung inflammation part, so that the excessive active oxygen aggregation is relieved, the excessive inflammatory reaction is relieved, and the lung inflammation treatment effect is improved. Therefore, pathological states such as oxidative stress and excessive inflammation of acute lung injury are effectively improved, and the traditional Chinese medicine composition has clinical transformation and application effects on treatment and popularization of inflammatory diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of the treatment of acute lung injury (ALI), and specifically relates to a preparation method of a macrophage membrane-modified MPDA@CeO 2 biomimetic nanocomposite and its therapeutic application in acute lung injury. Background Art

[0002] Acute lung injury (ALI) is a severe pulmonary inflammatory response that can rapidly progress to acute respiratory distress syndrome (ARDS). It is a severe and common pulmonary inflammatory response caused by various reasons, and its main feature is the damage of the alveolar-capillary membrane, resulting in a large amount of fluid and protein exudation between the alveoli and capillaries, posing a major threat to the health of patients. Despite the progress made in medical management, the mortality rate of ARDS remains high, and many survivors will suffer from long-term physical and cognitive dysfunction.

[0003] The etiology of ALI is complex and diverse, including direct factors such as inhalation of toxic substances, pulmonary infection or chest trauma, and indirect factors such as severe infection, shock or systemic inflammatory response syndrome, etc. Its main pathophysiological feature is the damage of the alveolar-capillary membrane, resulting in an increase in the permeability of pulmonary capillaries. This increased permeability causes protein-rich fluid to leak from the blood vessels into the alveoli, forming pulmonary edema, reducing the effective gas exchange surface, and leading to hypoxemia. The inflammatory response in the acute phase causes white blood cells (especially neutrophils) to accumulate in the lungs, releasing inflammatory mediators, causing further lung tissue damage. These inflammatory cells and mediators also damage pulmonary capillary endothelial cells and alveolar epithelial cells, exacerbating the destruction of the alveolar-capillary barrier. In addition, excessive reactive oxygen species also play an important role in the damage process, further damaging cell membranes, proteins and nucleic acids, exacerbating tissue damage and inflammatory response, and having a negative impact on lung function.

[0004] The treatment of ALI / ARDS faces a series of challenges and limitations. The current treatment still mainly focuses on supportive measures, and the high mortality rate still exists, especially in critically ill patients. Inflammation and cell damage play a key role in the onset and progression of the disease, but the methods for treating these processes are limited. In addition, there is a wide range of individual differences, and more individualized treatment methods are needed. Early diagnosis is difficult, resulting in possible treatment delays. Even if the patient survives, they may face long-term lung function damage and other complications, posing a long-term challenge to the quality of life and medical resources. Therefore, the treatment of ALI / ARDS is still a complex field that requires more in-depth research to develop more effective, specific and individualized treatment methods to improve the prognosis of patients.

[0005] The pathogenesis of ALI / ARDS is complex and involves multiple links such as the activation of inflammatory cells, oxidative stress, the excessive release of cytokines, and the destruction of the extracellular matrix. Among them, oxidative stress is considered a key driving factor in the development of ALI. The accumulation of excessive reactive oxygen species (including superoxide anion (O 2 · - ), hydroxyl radical (·OH), hydrogen peroxide (H 2 O 2 ), singlet oxygen ( 1 O 2 ) etc.) is an important factor leading to ALI, which can exacerbate the inflammatory response, resulting in lung tissue injury and dysfunction.

[0006] ROS is a mediator of the inflammatory response, which can activate the intracellular signal transduction pathway, increase the production and release of inflammatory cytokines, thus leading to the continuous development of the inflammatory response. A moderate inflammatory response plays a good defensive role in the human body. However, an excessive inflammatory response will cause harm to the body. Inflammation plays a core role in the pathogenesis of ALI. Excessive ROS causes imbalance between oxidation and antioxidant systems in the body, leading to oxidative stress, and then leading to upregulation of catabolic enzymes, degradation of the extracellular matrix, reduced matrix synthesis, lung inflammation, alveolar cell death and senescence, thus promoting the overall progression of ALI.

[0007] Therefore, developing new therapies that can effectively scavenge reactive oxygen species (ROS) and inhibit the inflammatory response is the key to treating ALI. Summary of the Invention

[0008] The purpose of the present invention is to provide the preparation and application of a macrophage membrane biomimetic M-MPDA@CeO 2 nano - composite, which can achieve active targeting of the inflammatory site, thereby reducing the accumulation of excessive reactive oxygen species, alleviating the excessive inflammatory response, and effectively improving the pathological state of ALI.

[0009] To achieve the above - mentioned purpose, the present invention adopts the following technical solutions:

[0010] A nano - composite material, named M-MPDA@CeO 2 , consists of a core composed of mesoporous polydopamine loaded with ultrasmall cerium dioxide particles and is wrapped by a macrophage cell membrane.

[0011] The preparation method of the nano - composite material includes the following steps:

[0012] (1) Preparation of the core: Add MPDA powder to absolute ethanol and stir. After mixing evenly, add CeO 2 nano - particles, and stir at room temperature; after centrifugation, wash, and vacuum freeze - dry to obtain MPDA@CeO 2 powder;

[0013] (2) Macrophage membrane encapsulation: Macrophage membrane microcapsules were encapsulated with MPDA@CeO 2 The core-water solution was fully mixed; the mixed solution was ultrasonically treated and then squeezed back and forth under a 200 nm porous membrane to obtain M-MPDA@CeO 2 Solution; M-MPDA@CeO 2 The solution was vacuum freeze-dried to obtain the nanocomposite material M-MPDA@CeO 2 .

[0014] Step (1) CeO 2 The nanoparticles were prepared by the following method:

[0015] Weigh 500 mg Ce(NO 3 ) 3 6H 2 CeO and 200 mg tri-n-octylphosphine oxide were placed in a beaker, 20 mL of anhydrous ethanol and 2 ml of deionized water were added, and the mixture was heated and stirred. When the solute was completely dissolved, 5 ml of octadecene and 100 μL of oleylamine were added, and the mixture was heated to 280 ° C for 1 hour under a continuous nitrogen protection environment. After cooling to room temperature, the mixture was washed with anhydrous ethanol, and the supernatant was discarded after centrifugation. Subsequently, the precipitate was added to an acetone solution for solvent exchange, and the supernatant was removed by centrifugation again and the mixture was opened and allowed to stand. The precipitate was vacuum freeze-dried to obtain a light yellow powder, which was CeO 2 Nanoparticles.

[0016] The MPDA powder is prepared by the following method:

[0017] 150 mg of dopamine hydrochloride and 100 mg of F127 were dissolved in 10 mL of deionized water and 10 mL of ethanol solution, respectively; after being fully dissolved, the above solutions were mixed, 160 μL of 1,3,5-trimethylbenzene was added to the mixed solution, and water bath sonication was performed; 375 μL of ammonia water was slowly added to the system under magnetic stirring conditions, and the mixed solution was reacted at 50° C. for 2 hours; after centrifugation, the precipitate was washed with ethanol and ultrapure water respectively; the final product was vacuum freeze-dried to obtain MPDA powder.

[0018] Step (1) The ratio of MPDA powder to anhydrous ethanol is 20 mg: 20 mL; CeO 2 The amount is 5mg.

[0019] The macrophage membrane microcapsules are prepared by the following method:

[0020] (1) At 37°C, 5% CO 2The RAW 264.7 macrophage cell line derived from mice was cultured in DMEM complete medium containing 10% serum in a constant temperature cell incubator; when the cell confluence rate reached 90%, the cells were gently washed 3 times with PBS; the cells were scraped off and transferred to a centrifuge tube, centrifuged to discard the supernatant, and the cell pellet at the bottom of the tube was collected;

[0021] (2) Cell membrane extraction: Add membrane protein extraction buffer to the centrifuge tube, pipette and mix well, and place on ice bath; collect the pretreated cell suspension into a pre-cooled cell homogenizer, homogenize slowly multiple times to fully lyse the cells; then collect the product of the previous step into a centrifuge tube, centrifuge, and collect the supernatant into another centrifuge tube; centrifuge again, and the precipitate obtained at the bottom of the tube is the macrophage cell membrane fragment; resuspend the macrophage cell membrane fragment in PBS, and use a liposome extruder to extrude it multiple times through a polycarbonate porous membrane with a pore size of 400 nm to prepare macrophage cell membrane microcapsules.

[0022] The mixing ratio of the macrophage cell membrane microcapsules and the MPDA@CeO 2 core aqueous solution is that the macrophage cell membrane microcapsules and the MPDA@CeO 2 core aqueous solution are in a mass ratio of 10:1.

[0023] The conditions for ultrasonic treatment of the mixed solution are to ultrasonicate for 5 min at a frequency of 30 kHz and a power of 100 W using an ultrasonic disperser.

[0024] The application of the nanocomposite in the preparation of drugs for treating ALI or ARDS caused by ALI.

[0025] The beneficial effects obtained by the present invention are: The present invention provides an M-MPDA@CeO 2 nanocomposite with inflammatory targeting and synergistic antioxidant functions. M-MPDA@CeO 2 not only has the advantages of an intelligent nanoplatform, can achieve active targeting of the inflammatory site, and controllably release CeO 2 at the inflammatory site, but also can achieve a synergistic effect with the MPDA carrier, thereby effectively improving the pathological state of ALI. It provides a new strategy for the treatment of ALI.

[0026] Experimental results show that the prepared M-MPDA@CeO 2It has catalase and superoxide dismutase activities, has no obvious toxicity to cells at therapeutic concentrations, has good biocompatibility in vivo, its blood half-life is 10.53 ± 1.61 h, it can significantly accumulate in the inflamed injury site, reduce LPS-induced lung injury in mice, alleviate pulmonary edema, reduce the expression of inflammatory factors, can effectively scavenge excessive ROS in MLE-12 cells caused by LPS, protect cells from oxidative damage, repair damaged mitochondria, and reduce apoptosis. M-MPDA@CeO 2 It can significantly target to the lung injury site in vivo and has excellent antioxidant function in vivo. Description of the Drawings

[0027] Figure 1 : Transmission electron microscope morphologies of different substances, where A is CeO 2 ; B is MPDA; C is MPDA@CeO 2 ; D is M-MPDA@CeO 2 .

[0028] Figure 2 Representative western blot analysis of Integrinβ1, CD47 and TNFα-2 in RAW264.7, macrophage cell membrane microcapsules and M-MPDA@CeO 2 .

[0029] Figure 3 (A) Nitrogen adsorption (desorption) isotherm (BET) of MPDA; (B) Pore size analysis of MPDA.

[0030] Figure 4 CeO 2 , MPDA@CeO 2 , M-MPDA@CeO 2 Particle size diagram and potential diagram; where Figure 4 D is the Zeta potential diagram of MPDA, CeO 2 , macrophage cell membrane microcapsules, M-MPDA@CeO 2 .

[0031] Figure 5 M-MPDA@CeO 2 Stability in different solutions.

[0032] Figure 6A For the X-ray photoelectron spectrum of C of M-MPDA@CeO 2 : Figure 6B For the X-ray photoelectron spectrum of O of M-MPDA@CeO 2 Figure 6C For the X-ray photoelectron spectrum of Ce of M-MPDA@CeO 2 .

[0033] Figure 7 CeO 2 , MPD, M-MPDA@CeO 2 UV absorption spectra

[0034] Figure 8 CeO 2 , MPD, M-MPDA@CeO 2 FT-IR spectra

[0035] Figure 9 M-MPDA@CeO 2 SEM morphology images of M-MPDA@CeO lysed in hydrogen peroxide solution and Ce release curves in hydrogen peroxide solution

[0036] Figure 10 M-MPDA@CeO 2 Analysis diagrams of antioxidant capacity and catalytic activity Figure 10 A, Figure 10 B are the antioxidant capacities of M-MPDA@CeO 2 for scavenging ·OH and O 2 · - , Figure 10 C is the CAT enzyme-mimicking activity of M-MPDA@CeO 2 for catalyzing hydrogen peroxide to generate oxygen, Figure 10 D is the UV-visible absorption spectra of M-MPDA@CeO 2 after incubation with TMB + ·OH at different concentration gradients

[0037] Figure 11 M-MPDA@CeO 2 Cytotoxicity to RAW264.7 cells and MLE-12 cells

[0038] Figure 12 M-MPDA@CeO 2 Effect on the viability of LPS-stimulated MLE-12 cells

[0039] Figure 13 Transmission electron microscope images of MLE-12 cells after different intervention treatments, (→ indicates mitochondria, scale bar: 500 nm).

[0040] Figure 14 Calcein-AM / PI double staining analysis of MLE-12 cells after different intervention treatments, where Figure 14 A is the Calcein-AM / PI double staining image of MLE-12 cells after different intervention treatments, scale bar 400 μm Figure 14 B is the fluorescence quantification image of Calcein-AM / PI double staining

[0041] Figure 15 Images of mitochondrial membrane potential changes in MLE-12 cells after different treatments, scale bar: 200 μm.

[0042] Figure 16A Fluorescence microscopy images of Cy5.5 uptake by MIE-12 cells co-incubated with M-MPDA@Cy5.5 solution (100 μg / mL) for different times, scale bar: 100 μm. Figure 16B Fluorescence quantification chart of MIE-12 cells' uptake of M-MPDA@Ce6 (***P<0.001); Figure 16C ICP-OES detection of Ce uptake by MIE-12 and M-MPDA@CeO 2 Ce uptake by cells co-incubated for different times.

[0043] Figure 17A Fluorescence microscopy images of ROS scavenging ability in MLE-12 cells under different treatment conditions, scale bar: 400 μm. Figure 17B Quantification image of DCFH-DA fluorescence intensity (n = 3, P<0.001 vs control group; *P<0.05 vs model group; ***P<0.001 vs model group; &P<0.05); Figure 17C Images of ROS scavenging ability in MLE-12 cells detected by flow cytometry under different treatment conditions.

[0044] Figure 18 Relative mRNA levels of TNF-α (I), IL-1β (II), and IL-6 (III) in LPS-induced RAW264.7 cells after different treatments (n = 3, P<0.001 vs control group; **P<0.01 vs model group; ***P<0.001 vs model group; &P<0.05; &&P<0.01). Figure 18 B Relative gene expression levels of iNOS (I), CD86 (II), Arg1 (III), and CD206 (VI) in RAW264.7 macrophages detected by qRT-PCR, (n = 3, **P<0.01 vs model group; ***P<0.001 vs model group).

[0045] Figure 19 M-MPDA@CeO 2 Results of hemolytic activity detection.

[0046] Figure 20 M-MPDA@CeO 2Detection results of blood WBC (a), RBC (b), PLT (c), HGB (d), ALT (e), AST (f), BUN (g), and Cr (h) in mice 2 weeks after injection compared with the control group.

[0047] Figure 21 M-MPDA@CeO 2 HE detection results of major organs in mice 2 weeks after injection compared with the control group, scale bar: 200 μm.

[0048] Figure 22 Body weight change curves of mice with different treatments.

[0049] Figure 23A In vivo fluorescence imaging of ALI mice after tail vein injection of MPDA@Cy5.5 or M-MPDA@Cy5.5 at different times; Figure 23B Fluorescence images of major organs heart, liver, spleen, lung, and kidney in ALI mice 24 hours after tail vein injection of MPDA@Cy5.5 or M-MPDA@Cy5.5 nanomaterials; Figure 23C Quantification of fluorescence intensity of major organs heart, liver, spleen, lung, and kidney in ALI mice.

[0050] Figure 24 Intravenous injection of M-MPDA@CeO 2 (5 mg / kg) into normal mice (n = 3) and the plasma concentration-time curve of Ce.

[0051] Figure 25 Total number of cells in bronchoalveolar lavage fluid. (n = 3, P < 0.001 vs control group; ***P < 0.001 vs model group; &&P < 0.01; ns no significant difference).

[0052] Figure 26 Total protein concentration in bronchoalveolar lavage fluid. (n = 3, P < 0.001 vs control group; *P < 0.05 vs model group; ***P < 0.001 vs model group; &&&P < 0.001).

[0053] Figure 27 Wet / dry ratio of lung lobes. (n = 3, P < 0.001 vs control group; *P < 0.05 vs model group; ***P < 0.001 vs model group; &P < 0.05; ns no significant difference).

[0054] Figure 28A ELISA detection of TNF-α in bronchoalveolar lavage fluid of mice in different treatment groups. Figure 28B ELISA detection of IL-1β content in bronchoalveolar lavage fluid of mice in different treatment groups. Figure 28CThe IL-6 content in the bronchoalveolar lavage fluid of mice in different treatment groups was detected by ELISA. One-way ANOVA was used for statistical comparison. (n = 3, P < 0.001 vs. the control group; *P < 0.05 vs. the model group; **P < 0.01 vs. the model group; ***P < 0.001 vs. the model group; &P < 0.05; &&P < 0.01; &&&P < 0.001; ns indicates no significant difference).

[0055] Figure 29 Survival curves of mice in the M-MPDA@CeO2 intervention group and the control group.

[0056] Figure 30 Gross view of the lung tissue of mice in each treatment group and HE staining of lung lobes, scale bar: 200 μm.

[0057] Figure 31 Tunel staining of the lung tissue of mice in each treatment group, scale bar: 200 μm.

[0058] Figure 32 MDA immunohistochemical staining of the lung tissue of mice in each treatment group, scale bar: 200 μm.

[0059] Figure 33 Reactive oxygen species staining of the lung tissue of mice in each treatment group, scale bar: 200 μm. Detailed implementation methods

[0060] Synthesis of CeO 2

[0061] Accurately weigh 500 mg of Ce(NO 3 ) 3 ·6H 2 O and 200 mg of trioctylphosphine oxide in a 50 mL beaker, add 20 mL of absolute ethanol and 2 mL of deionized water, heat and stir to mix evenly. When the solute is completely dissolved, transfer it to a 20 mL glass flask, add 5 mL of octadecene and 100 μL of oleylamine, and heat to 280 °C under a continuous nitrogen protection environment for 1 hour. The color of the mixture finally turns into a pale yellow colloidal solution. After cooling to room temperature, wash with absolute ethanol, centrifuge and discard the supernatant, and wash 3 times. Subsequently, add the precipitate to acetone solution for solvent exchange, centrifuge again to remove the supernatant and leave it open to stand for 2 hours to promote the volatilization of residual acetone, which helps to remove the residual organic solvents. Vacuum freeze-dry the precipitate to obtain a pale yellow powder and store it at -20 °C for later use.

[0062] Synthesis of MPDA

[0063] ​Dissolve 150 mg of dopamine hydrochloride (DA) and 100 mg of F127 in 10 mL of deionized water and 10 mL of ethanol solution respectively. After complete dissolution, mix the above solutions, add 160 μL of 1,3,5-trimethylbenzene (TMB) to the mixed solution, and perform ultrasonic bath for 6 minutes. Slowly add 375 μL of ammonia water to the system under magnetic stirring conditions, and react the mixed solution at 50 °C for 2 hours. Centrifuge at 9000 rpm / min for 10 minutes, and wash the precipitate with ethanol and ultrapure water three times each. The final product is stored at -20 °C after vacuum freeze-drying for later use.

[0064] MPDA@CeO 2 Synthesis

[0065] Weigh 20 mg of MPDA powder, add it to 20 mL of anhydrous ethanol and stir. After mixing evenly, add 5 mg of CeO 2 , and stir magnetically at room temperature for 12 hours. Then centrifuge at 9000 r / min for 10 minutes, wash three times with anhydrous ethanol, and the centrifuged product is vacuum freeze-dried to obtain MPDA@CeO 2 powder.

[0066] Macrophage cell membrane extraction

[0067] (1) Cell collection: Culture the mouse-derived RAW 264.7 macrophage cell line in a constant temperature cell incubator (37 °C, 5% CO 2 ) using DMEM complete medium containing 10% serum. When the cell confluence rate reaches 90%, gently wash the cells three times with PBS. Scrape the cells with a cell scraper and transfer them to a 1.5 ml centrifuge tube. The cell density is about 2×10 7 cells / mL, centrifuge at 1000 rpm, 4 °C for 5 minutes, discard the supernatant, and collect the cell precipitate at the bottom of the tube.

[0068] (2) Cell membrane extraction: Add membrane protein extraction buffer to the centrifuge tube, pipette and mix evenly, and incubate on ice for 15 min; collect the pretreated cell suspension into a pre-cooled cell homogenizer, and homogenize slowly and evenly 30 times at 4 °C to fully lyse the cells. Then collect the product of the previous step into a centrifuge tube, centrifuge at 2000 rpm / min for 10 minutes at 4 °C, and collect the supernatant into another centrifuge tube. Centrifuge at 14000 rpm / min for 30 minutes at 4 °C, and the precipitate obtained at the bottom of the tube is macrophage cell membrane fragments. Resuspend the macrophage cell membrane fragments in PBS, and use a liposome extruder to extrude through a polycarbonate porous membrane with a pore size of 400 nm for 10 - 15 times to prepare macrophage cell membrane microcapsules (MM NV), and store the obtained macrophage cell membrane microcapsules in a 4 °C refrigerator for later use.

[0069] M-MPDA@CeO2 Synthesis of nano - composite

[0070] Co - extrusion was used to coat the macrophage cell membrane on the surface of MPDA@CeO 2 The extracted macrophage cell membrane microcapsules were fully mixed with the MPDA@CeO 2 core aqueous solution at a mass ratio of 10:1. The mixture was sonicated for 5 min using an ultrasonic disperser at a frequency of 30 kHz and a power of 100 W, and then extruded back and forth 15 times through a 200 - nm polycarbonate porous membrane using a liposome extruder. The prepared M - MPDA@CeO 2 solution was vacuum freeze - dried and stored at 4 °C for later use.

[0071] Synthesis of fluorescently labeled nano - composite

[0072] Preparation of nano - composite loaded with Cy5.5 (excitation = 683 nm, emission = 703 nm) fluorescence. Except for the difference in the core, that is, MPDA@Cy5.5, the remaining steps were the same, which was used for fluorescence test evaluation in in vitro and in vivo experiments.

[0073] Protein immunoblotting to verify the surface - characteristic proteins of M - MPDA@CeO 2 Surface - characteristic proteins

[0074] To confirm that M - MPDA@CeO 2 can maintain the protein composition on the original macrophage cell membrane and verify the successful coating of the macrophage cell membrane on the surface of MPDA@CeO 2 surface, the Integrinβ1, CD47, and TNFα - 2 proteins of macrophages, macrophage cell membrane microcapsules, and M - MPDA@CeO 2 were detected by Western blotting (WB). The presence of key proteins on the surface macrophage cell membrane of M - MPDA@CeO 2 was used to verify the successful coating of the macrophage cell membrane on the surface of MPDA@CeO 2 surface.

[0075] (1) Protein extraction: Prepare the lysis buffer and add PMSF so that the final concentration of PMSF is 1 mM. Mix well to obtain the working lysis buffer. After centrifuging and precipitating the sample to be tested, add 250 μL of the lysis buffer to each tube of the sample, and mix well by pipetting several times. Place it on ice for 5 - 10 minutes for lysis, and shake the centrifuge tube back and forth several times during this period to ensure complete lysis. After lysis, centrifuge the sample and pipette the supernatant into a 1.5 - mL centrifuge tube, and quickly freeze and store for later use.

[0076] (2) Protein quantification: Use a BCA protein quantification kit to quantify the proteins in the samples and measure according to the kit instructions. According to the quantification results, use PBS buffer to adjust the protein concentrations of the samples to be electrophoresed together to be consistent. Add protein loading buffer (5×) to the samples at a ratio of 4:1 (v / v), mix well, and heat in a 100 °C metal bath for 4 minutes to fully denature the proteins.

[0077] (3) SDS-PAGE gel electrophoresis: Prepare a 12% polyacrylamide separating gel using an SDS-PAGE gel rapid preparation kit. After the separating gel solidifies, remove the moisture and blot it dry with absorbent paper. Prepare the upper 5% stacking gel according to the instructions. After the stacking gel solidifies, place it in the electrophoresis tank. Remove the comb from the gel and slowly add the samples to the gel loading wells using a loading needle, loading 10 μL per well. First, run the gel at 70 V for 0.5 hours, and then run the gel at 140 V for 1 hour to fully separate the proteins.

[0078] (4) Transfer: Take a PVDF membrane of the same size as the separating gel, soak it in methanol for 5 minutes to activate it, and then soak it in the pre-prepared transfer buffer. Remove the protein separating gel after electrophoresis, remove the stacking gel, wash it several times with distilled water, and then soak it in the transfer buffer. Place the protein separating gel on multiple layers of filter paper, and then place the PVDF membrane on the protein separating gel. Ensure that there are no bubbles on the contact surface between the PVDF membrane and the protein separating gel. Cover the PVDF membrane with multiple layers of filter paper soaked in the transfer buffer, clamp it, place it in the electrophoresis tank, add enough transfer buffer, and transfer at 40 V for 1.5 hours.

[0079] (5) Blocking: After the transfer is completed, take out the PVDF membrane, keep it moist, rinse it 3 times with TBST, and then place the PVDF membrane in 5% skim milk powder prepared with TBST for blocking for 2 hours.

[0080] (6) Incubate with primary antibody: Dilute the primary antibodies (Anti Integrinβ1, Anti CD47, Anti TNFα-2) to the working concentration with TBST according to the usage method in the instructions. Place the PVDF membrane in an incubation box, add the diluted primary antibodies to submerge the PVDF membrane. Place the incubation box in a 4 °C refrigerator and incubate overnight. The next day, take out the PVDF membrane, place it in TBST, and rinse it 3 times on a shaker, 10 minutes each time.

[0081] (7) Incubate with secondary antibody: Put the PVDF membrane rinsed with TBST back into the incubation box, add the horseradish peroxidase-labeled secondary antibody diluted with TBST to submerge the PVDF membrane, and incubate at room temperature for 1 hour.

[0082] (8) Color development: After incubating the secondary antibody, place the PVDF membrane in TBST and rinse it on a shaker 3 times for 10 minutes each. Develop the color with a protein developing solution, prepared according to the instructions, then drop it on the PVDF membrane, and finally expose and develop it in an imaging system.

[0083] Characterization of Materials

[0084] Transmission Electron Microscope

[0085] The Transmission Electron Microscope (TEM) is a high-resolution microscope that uses an electron beam instead of light to form a high-resolution image of a sample. Compared with optical microscopes, TEM can provide higher spatial resolution, allowing clear microscopic morphology and more detailed structures to be observed, and is often used to analyze and reveal crystal structures. The TEM forms an image through the electron beam transmitted through the sample. The electron beam penetrates the sample and is then focused into a beam of electrons passing through the sample. By measuring the scattering pattern of the electrons, a high-resolution two-dimensional projection image is formed. In this article, the morphology of CeO 2 、M-MPDA@CeO 2 was tested using a JEM 1200EX tungsten filament transmission electron microscope with an accelerating voltage of 120 kV. An appropriate amount of the dispersed sample was dropped on a copper mesh with an ultra-thin carbon film for sample preparation, dried at room temperature, and then observed for morphology on the machine.

[0086] Scanning Electron Microscope

[0087] The Scanning Electron Microscope (SEM) is a general technique for imaging nanoscale samples. The SEM uses an electron gun to generate a high-energy electron beam, which is focused and scanned across the surface of the sample at high speed. When the electron beam interacts with the sample surface, secondary electrons and reflected electrons are generated. By measuring the signals of these secondary electrons and reflected electrons, a high-resolution image of the sample surface is formed. In this article, the morphology of MPDA and MPDA@CeO 2 was tested using a Sigma300 scanning electron microscope with an accelerating voltage of 5 kV. An aqueous solution of the nanoparticles was dropped on a silicon wafer for sample preparation. After drying, the sample was mounted on the SEM sample holder, ensuring that the sample was aligned with the path of the electron beam, and operated in a low-vacuum environment, and then observed for morphology on the machine.

[0088] Nitrogen Adsorption (Desorption) Isotherm of MPDA

[0089] The nitrogen adsorption (desorption) isotherm (BET) is an experimental measurement technique used to study the pore structure and surface area of materials. This technique typically uses nitrogen as the adsorbent and measures the amount of nitrogen adsorbed onto the material surface at different relative pressures to obtain information about the pore structure and specific surface area. An appropriate amount of MPDA powder is pretreated, such as degassing, to remove the gas adsorbed on the sample surface and in the pores. Then, nitrogen adsorption experiments are carried out at different relative pressures, and the adsorption amounts are recorded. Finally, by applying the BET (Brunauer - Emmett - Teller) and BJH (Barrett - Joyner - Halenda) theories to analyze the adsorption data, the specific surface area, pore diameter, etc. of the sample are calculated.

[0090] Determination of potential and particle size

[0091] Potential particle size detection is a method for obtaining particle size and Zeta potential by measuring the movement of particles in solution through dynamic light scattering (DLS) technology. CeO 2 , MPDA, macrophage cell membrane microcapsules, M - MPDA@CeO 2 are added to different potential or particle size measurement cells. Ensure that the samples are fully and evenly suspended before the experiment starts to avoid particle aggregation. Start the instrument and perform necessary system calibrations. Check the light source, detector, and other key components of the instrument to ensure that the instrument is in normal working condition. Put the cell of each sample into the instrument one by one, set the measurement parameters, and record the data. To improve the accuracy of the experimental results, repeat the measurement 3 times.

[0092] M - MPDA@CeO 2 Stability test

[0093] To evaluate the stability of M - MPDA@CeO 2 , 2 mg of M - MPDA@CeO 2 is dissolved into 5 ml of DMEM, FBS, and PBS solutions. After standing for one week at a constant temperature of 37 °C, an appropriate amount is taken out, and a particle size analyzer is used to detect the effect of different solutions on the particle size of M - MPDA@CeO 2 .

[0094] X - ray photoelectron spectroscopy analysis

[0095] X - ray photoelectron spectroscopy (XPS) is a technique used to analyze the chemical state and composition of elements on the surface of substances. XPS uses incident X - rays to excite the electrons on the surface of substances, causing these electrons to break away from the atoms and become photoelectrons. Measuring the energy and intensity of these photoelectrons can provide information about the surface elements. M - MPDA@CeO 2The sample was tested. The spectrometer was equipped with a monochromatic aluminum Kα X-ray source (1486.6 eV). X-rays were generated by the excitation source and focused onto the sample surface. The photoelectron signals detached from the sample were collected, and the acquired photoelectron spectroscopy was analyzed.

[0096] Ultraviolet absorption spectroscopy

[0097] Ultraviolet-Visible Spectroscopy (UV-Vis) is an analytical method used to study the absorption characteristics of substances in the ultraviolet and visible light regions. When electrons in a substance are excited from a low energy level to a high energy level, the absorption spectrum exhibits characteristic absorption bands. The position and intensity of the absorption bands provide information about the molecular structure, concentration, and chemical state of the substance. CeO 2 , MPDA, and M-MPDA@CeO 2 were diluted to a certain concentration with ultrapure water and ultrasonically dispersed thoroughly. 3 mL of each sample was added to a quartz cuvette, and the ultraviolet absorption spectra of the samples to be measured were detected using a UV-visible spectrophotometer.

[0098] Fourier transform infrared spectroscopy detection

[0099] Fourier Transform Infrared Spectroscopy (FT-IR) is an advanced infrared spectroscopy technique that uses the Fourier transform principle and has the characteristics of high sensitivity and high resolution by rapidly acquiring the entire spectrum. A small amount of completely dried CeO 2 , MPDA, and M-MPDA@CeO 2 powders were added to an appropriate amount of KBr powder, ground thoroughly, and pressed into tablets using a tablet press. The infrared absorption spectra of the samples to be measured were detected using an FT-IR spectrometer respectively.

[0100] M-MPDA@CeO 2 Release test of CeO 2 in

[0101] The release of CeO 2 depends on the cleavage of the carrier structure. First, the morphological image of M-MPDA@CeO 2 after cleavage was observed by SEM after treatment with H 2 O 2 (1 mM) for 2 h. To understand in detail the release amount of CeO 2 at each time point in the simulated inflammatory environment, first, 1 mg of M-MPDA@CeO 2 powder was weighed, and aqua regia (concentrated HNO 3 / Concentrated HCl, v / v = 1:3) was made up to 5 mL and digested overnight, and the content of Ce in 1 mg M-MPDA@CeO was determined by inductively coupled plasma optical emission spectrometry (ICP-OES). 2 The content of Ce in M-MPDA@CeO (n = 3) was then determined. Another 5 mg of M-MPDA@CeO powder was separately dispersed in 10 mL of PBS solution containing H 2 O 2 (1 mM) and PBS solution without H 2 O 2 O 2 . Then the solution was placed on a shaker at 37 °C and oscillated at a certain frequency to achieve the slow release of CeO 2 . At different time points, 1 mL of the solution was taken out, centrifuged, and the supernatant was taken. At the same time, after each time the solution was taken out, an equal volume of PBS buffer was added to the original solution and the oscillation was continued. The supernatant obtained at each time point was also made up to 5 mL with aqua regia and digested overnight, and the content of Ce in the supernatant at each time point was determined by ICP-OES (n = 3), and the release curve was obtained accordingly.

[0102] Detection of the ability of M-MPDA@CeO 2 to scavenge reactive oxygen species

[0103] Reactive oxygen species (ROS) refer to a class of highly reactive oxidizing molecules or ions produced in living organisms, including O 2 · - , ·OH, H 2 O 2 etc. Excessive reactive oxygen species can cause lipid peroxidation and produce reactive lipid dicarbonyls. These lipid oxidation products may be the most significant mediators of oxidative damage, and they have a strong oxidative effect on molecules in living organisms, causing oxidative damage to biological macromolecules such as cell membranes, proteins, and nucleic acids.

[0104] (1) Electron paramagnetic resonance: Electron paramagnetic resonance (EPR) is based on the resonance absorption of unpaired electrons in an external magnetic field. By microwave radiation, the unpaired electrons are excited from the low energy level to the high energy level to produce a resonance absorption signal, thus directly detecting the characterization method of paramagnetic substances containing unpaired electrons. The antioxidant ability of M-MPDA@CeO 2 to scavenge ·OH and O 2 · - was determined by the ESR method. An appropriate amount of M-MPDA@CeO 2 was mixed with PBS solution containing 1 mM H 2 O 2The system interacts, and DMPO / BMPO is added as a scavenger. DMPO / BMPO reacts with the generated free radicals to form free radical adducts. Next, the EPR signals of the sample at different reaction times are measured by an EPR instrument under appropriate experimental conditions, and the resonance absorption spectra of the generated free radical adducts are recorded.

[0105] (2) Catalase activity assay: Catalase (CAT) can catalyze H 2 O 2 to generate O 2 , and the catalytic ability of CAT can be evaluated by detecting the generation amount of O 2 . To understand the CAT activity of M-MPDA@CeO 2 , a dissolved oxygen meter is used to detect the content of O 2 generated by its catalysis of hydrogen peroxide. First, solutions of different concentrations of M-MPDA@CeO 2 (50 μg / ml, 100 μg / ml, 150 μg / ml, 200 μg / ml) are prepared, and then 1 ml of each is added to 1 mM H 2 O 2 solution to make the total system 5 mL. The solution is slowly and evenly stirred with a glass rod, and a dissolved oxygen meter is used to detect the generated O 2 . Oxygen is measured every 1 min for a total duration of 15 min.

[0106] (3) TMB color reaction: The TMB color reaction is a method commonly used to detect hydroxyl radicals. The hydroxyl radical scavenging ability of M-MPDA@CeO 2 is investigated by the color reaction of the TMB / H 2 O 2 system. 100 μL of solutions of different concentrations of M-MPDA@CeO 2 (50 μg / ml, 100 μg / ml, 150 μg / ml, 200 μg / ml, 250 μg / ml), 3 mL of 1 mM H 2 O 2 solution, and 100 μL of TMB (0.8 mmol / L) are mixed in sequence and reacted in a 37 °C water bath for 5 min. The ultraviolet-visible absorption spectrum of the reacted solution is measured in the wavelength range of 550 - 750 nm.

[0107] Statistical analysis

[0108] All experimental data were statistically analyzed using Origin 2021 software. The data were expressed as mean ± standard deviation. The t-test was used for comparison between two samples, and one-way analysis of variance (ANOVA) was used for comparison of multiple groups of data. P<0.05 indicated that the difference was statistically significant.

[0109] Results

[0110] Morphology analysis

[0111] Transmission electron microscope images showed that the synthesized CeO 2 was uniformly dispersed ultra-small dot-like nanoparticles with a particle size of about 5 nm ( Figure 1 A); MPDA presented as a spherical structure with uniform distribution and uniform size, and the particle size was about 200 nm ( Figure 1 B). After loading CeO 2 , MPDA@CeO 2 still presented as a sphere with uniform size and had good dispersibility. The adsorption of CeO 2 could be clearly seen on the outer surface of MPDA, indicating that CeO 2 could be loaded into the MPDA structure ( Figure 1 C). To verify that the macrophage cell membrane was successfully coated on MPDA@CeO 2 , the microscopic morphology of M-MPDA@CeO 2 was first observed by transmission electron microscopy. The results showed that a significant single-layer light coating could be seen on the outer surface of M-MPDA@CeO 2 , with clear boundaries, showing an obvious core-shell structure, presumably the outer-coated macrophage cell membrane ( Figure 1 D).

[0112] Verification of marker proteins

[0113] Since macrophages achieve the homing effect on the inflammatory site through the functional proteins on the membrane, Western blot was used to detect the expression of important functional proteins Integrinβ1, CD47, and TNFα-2 on RAW264.7, macrophage cell membrane microcapsules, and M-MPDA@CeO 2 ( Figure 2 ). The results showed that compared with RAW264.7, both macrophage cell membrane microcapsules and M-MPDA@CeO 2 contained the above-mentioned functional proteins expressed by RAW264.7, indicating that the membrane proteins still existed after physical extrusion. GAPDH is a typical cytoplasmic secreted protein and was not expressed in macrophage cell membrane microcapsules and M-MPDA@CeO 2 , indicating that the extracted were pure macrophage cell membrane microcapsules.

[0114] Specific surface area and pore size

[0115] As shown in Figure 3, from the nitrogen adsorption (desorption) isotherm of MPDA, it can be seen that the specific surface area of MPDA calculated by the BET method is 1219.57 m 2 / g, and the pore size calculated by the BJH method is about 12.90 nm. The large specific surface area and pore structure of MPDA indicate its good potential for loading small molecule substances and can be used as a carrier for drugs and the like.

[0116] Determination of particle size and Zeta potential

[0117] CeO 2 、MPDA、M-MPDA@CeO 2 The particle size distributions are as Figure 4 shown in A - C. The results show that the average particle size of CeO 2 is about 4.85 ± 0.24 nm, the average particle size of MPDA is about 165.19 ± 2.14 nm, and the average particle size of M-MPDA@CeO 2 is about 217.78.19 ± 2.04 nm. By comparison, it can be seen that after being wrapped with macrophage cell membrane, the particle size of M-MPDA@CeO 2 increases significantly. The Zeta potential results are as Figure 4 shown in D. The Zeta potential of CeO 2 is 31.40 ± 5.61 mV, the Zeta potential of MPDA is -33.77 ± 7.73 mV, the Zeta potential of macrophage cell membrane microcapsules is -18.63 ± 5.89 mV, and the Zeta potential of M-MPDA@CeO 2 is -13.51 ± 4.87 mV. Through the analysis of the Zeta potential, it can be known that after coating with macrophage cell membrane, the positive charge carried by CeO 2 is shielded, and M-MPDA@CeO 2 shows a potential close to that of the macrophage cell membrane. The results indicate that the macrophage cell membrane has been successfully coated on the outer surface of M-MPDA@CeO 2 . The macrophage cell membrane camouflage helps M-MPDA@CeO 2 simulate the function of macrophages and lay a foundation for subsequent experiments.

[0118] Stability

[0119] In biomedicine, the stability of nanomaterials is of great significance for ensuring the sustainability, safety, and performance superiority of their applications. The stability of M-MPDA@CeO 2 was preliminarily evaluated by detecting the influence of different solutions on its particle size, as Figure 5As shown, we observed that M-MPDA@CeO 2 After standing in DMEM and PBS for one week, its particle sizes were measured to be 222.79±8.34 nm and 220.01±7.13 nm respectively, and the change in particle size was not significant, indicating good stability. The particle size in FBS was 200.80±6.17 nm, slightly smaller than that in DMEM and PBS but with little overall difference. This may be due to the interaction of the complex components of serum proteins with the surface of M-MPDA@CeO 2 , resulting in a change in surface charge. This change in charge may affect the interaction force between nanoparticles, making the measured particle size slightly smaller in FBS.

[0120] XPS analysis

[0121] As Figure 6A shown in -C, characteristic peaks of C and O appeared at the corresponding binding energy peaks of C1s and O1s for M-MPDA@CeO 2 , proving the presence of C and O elements in the material. In the Ce3d spectrum of M-MPDA@CeO 2 , Ce exists in both Ce 3+ (880.84, 885.16, 897.16, and 903.88 eV) and Ce 4+ (882.56, 889.13, 898.43, 901.13, 908.03, and 916.82 eV). The characteristic element peaks measured by XPS confirmed the presence of elements such as C, O, and Ce in the final product M-MPDA@CeO 2 , and also proved the existence of two valence states of Ce element, where Ce 3+ is the reduced state and Ce 4+ is the oxidized state. Since the enzyme-like activity of CeO 2 depends on the ratio of Ce 3+ and Ce 4 + , we further quantified this ratio. In M-MPDA@CeO 2 , the ratio of Ce 3+ :Ce 4+ =21.5 / 78.49. Ce 3+ is the reduction active site in CeO 2 , which can absorb, store, and release oxygen. Its redox cycle enables CeO 2 to absorb oxygen and form Ce 4+ , while under reducing conditions, Ce 4+ can be reduced to Ce 3+ . In addition, the presence of oxygen vacancies in CeO 2 enhances its surface activity.

[0122] Ultraviolet and infrared spectroscopy analysis

[0123] To verify the successful synthesis of M-MPDA@CeO 2 UV-Vis and FT-IR were used for characterization respectively. The results of the ultraviolet absorption spectrum are as Figure 7 shown. CeO 2 showed an absorption peak at 251 nm, and MPDA had a typical characteristic absorption peak at 280 nm, which corresponded to the π-π electronic transition of the aromatic ring in dopamine. The final product M-MPDA@CeO 2 showed the same characteristic absorption peak near 250 nm as CeO 2 . Generally speaking, the ultraviolet absorption peak of CeO 2 usually appears at a shorter wavelength, that is, in the ultraviolet light region. In the spectrum of CeO 2 , the specific absorption peak position and shape may be affected by factors such as the crystal structure, crystal morphology, and the presence or absence of oxygen vacancies of CeO 2 .

[0124] In addition, qualitative FT-IR structural analysis was carried out on the samples, and the results are as Figure 8 shown: The infrared spectrum results of CeO 2 showed that 1629 cm -1 was the O-H bending vibration peak of water, 1472 cm -1 , 1046 cm -1 were the peaks introduced by other impurities in the CeO 2 sample (C-O stretching vibration in the solvent), and 471 cm -1 was the Ce-O stretching vibration peak (characteristic peak) of CeO 2 . In the infrared spectrum of MPDA, 3390 cm -1 was the O-H bending vibration peak of water, 3230 cm -1 was the N-H stretching vibration peak, 3038 cm -1 was the =C-H stretching vibration peak of the aromatic ring, 2923 cm -1 was the C-H stretching vibration peak of methylene, 1611 cm -1 , 1492 cm -1 were the C=C double bond stretching vibration peaks of the aromatic ring, 1445 cm -1 was the out-of-plane bending vibration peak of C-H of methylene and methyl, 1350 cm -1 was the C-N stretching vibration peak of aromatic amine, 1285 cm -1 was the C-O stretching vibration peak of phenol, 1197 cm -1 was the C-N stretching vibration peak of aliphatic amine. M-MPDA@CeO 2In the infrared spectrum, 3396 cm -1 is the O-H bending vibration peak of water, 3226 cm -1 is the N-H stretching vibration peak, 3040 cm -1 is the =C-H stretching vibration peak of the aromatic ring, 2913 cm -1 is the C-H stretching vibration peak of methylene, 1598 cm -1 、1502 cm -1 is the C=C double bond stretching vibration peak of the aromatic ring, 1435 cm -1 is the out-of-plane bending vibration peak of C-H of methylene, 1350 cm -1 is the C-N stretching vibration peak of aromatic amine, 1285 cm -1 is the C-O stretching vibration peak of phenol, 1168 cm -1 is the P=O stretching vibration peak of phospholipids on the cell membrane, 1085 cm -1 is the C-O stretching vibration peak of esters on the cell membrane, 462 cm -1 is the Ce-O stretching vibration peak of CeO 2 . The above analysis results indicate the successful synthesis of M-MPDA@CeO 2 .

[0125] Release of CeO 2

[0126] SEM images show that after treatment with 1 mM H 2 O 2 for 2 h, M-MPDA@CeO 2 has obvious morphological lysis ( Figure 9 A). The disintegration characteristics of M-MPDA@CeO 2 in H 2 O 2 result in the responsive release of CeO 2 . The ICP-OES detection results show that the content of Ce element in M-MPDA@CeO 2 is 6.34%. The cumulative release rate of Ce in 1 mM hydrogen peroxide solution for 72 h is 73.41%, while in PBS solution, the cumulative release rate for 72 h is 9.79% ( Figure 9 B). The morphological lysis of M-MPDA@CeO 2 and the release of CeO 2 under hydrogen peroxide conditions indicate that the nanocomposite has high sensitivity to reactive oxygen species and controllable release characteristics.

[0127] Determination of free radical scavenging ability

[0128] To systematically study M-MPDA@CeO 2 ​Powerful reactive oxygen species scavenging ability. We selected representative reactive oxygen species: ·OH, O 2 · - and H 2 O 2 . Using DMPO to capture ·OH to form the DMPO / ·OH spin adduct, compared with the control group without M-MPDA@CeO 2 , after adding M-MPDA@CeO 2 , the ESR signal intensity decreased with the extension of reaction time. At 10 min of reaction, M-MPDA@CeO 2 could almost completely scavenge ·OH( Figure 10 A). This result verified that M-MPDA@CeO 2 had a strong ·OH scavenging ability. Using BMPO as the spin trap for superoxide, the results showed that a strong ESR signal was generated in the control group. After adding M-MPDA@CeO 2 , the signal intensity of ESR also decreased significantly with the extension of reaction time, proving its antioxidant ability to scavenge superoxide( Figure 10 B). After the reaction of M-MPDA@CeO 2 with H 2 O 2 , with the increase of the concentration of M-MPDA@CeO 2 and the extension of reaction time, the generated amount of O 2 also gradually increased( Figure 10 C), confirming that M-MPDA@CeO 2 had good CAT catalytic performance.

[0129] The ability of M-MPDA@CeO 2 to scavenge oxygen free radicals was evaluated by the TMB color reaction. In the TMB-H 2 O 2 reaction system, a typical color reaction and a characteristic absorption peak at 652 nm were rapidly generated. When M-MPDA@CeO 2 was added to the reaction system to interact, the blue reaction of TMB gradually faded, and the absorbance of the characteristic absorption peak at 652 nm decreased gradually with the increase of the concentration of M-MPDA@CeO 2 ( Figure 10 D). It showed that M-MPDA@CeO 2 could significantly scavenge oxygen free radicals, thus inhibiting the oxidation of TMB, and its scavenging ability was closely related to the concentration.

[0130] Evaluation of the in vitro biological effects of M-MPDA@CeO 2 nanocomposites

[0131] Turn on the ultraviolet lamp of the cell operation table half an hour in advance for irradiation sterilization, and preheat the complete DMEM medium in a 37°C water bath for standby. Take out the cryopreserved cells from the -80°C ultra-low temperature refrigerator and quickly place them in a 37°C constant temperature water bath to melt. Add the melted cell suspension to a centrifuge tube containing 3 mL of complete DMEM medium, centrifuge at 1000 rpm / min for 5 min, remove the medium, add 5 mL of DMEM cell medium to resuspend, and then transfer the cell suspension to a T25 cell culture flask and place it at 37°C, 5% CO 2 Cultivate in a cell incubator.

[0132] When the cell fusion rate reaches about 70-80%, perform cell passage. Take out the cell culture flask from the incubator, gently wash the cells with PBS 2 times, add 1 mL of 0.25% trypsin (preheated in a 37°C water bath) to digest the cells for 2 minutes, then add 3 mL of fresh complete medium to terminate the digestion, transfer to a 15 mL centrifuge tube, centrifuge at 1000 rpm / min for 5 min, remove the old medium, add 3 mL of fresh medium to resuspend, take 1 mL and add it to the cell culture flask, then add 3 mL of cell medium and mix well. Put the culture flask back into the 37°C, 5% CO 2 Cell incubator.

[0133] Digest, centrifuge and count the cells with good growth status by trypsin. Then, use the cell cryopreservation solution prepared with 50% medium, 40% FBS and 10% DMSO, aliquot it into cell cryopreservation tubes and label information such as cell name and cryopreservation date. Put the cryopreservation tubes into a programmed gradient cooling box, and then place the whole in a -80°C ultra-low temperature refrigerator to ensure the quality of cell cryopreservation and long-term storage.

[0134] M-MPDA@CeO 2 Cytotoxicity detection of MLE-12 and RAW264.7 cells

[0135] Seed the digested and resuspended cells in a 96-well plate at a density of 1×10 4 cells per well. After culturing overnight, discard the original medium. Add 100 μL of fresh medium containing 0, 12.5, 25, 50, 100, 200 μg mL -1 containing M-MPDA@CeO 2 to each well, and continue to culture for 24 hours. Then, discard the old medium and wash once with PBS. Subsequently, add 100 μL of serum-free medium containing 10% CCK-8 and incubate at 37°C for 1 hour. Finally, measure the absorbance value at a wavelength of 450 nm to calculate the cell viability.

[0136] M-MPDA@CeO 2 Protect MLE-12 cells from oxidative stress damage

[0137] CCK-8

[0138] MLE-12 cells were cultured at 1×10 4 The cells were seeded at a density of 100 μL per well in a 96-well plate. After overnight culture, the original culture medium was discarded and fresh culture medium containing 1 μg / ml LPS was added to each well to construct a cell oxidative damage model. After culturing for 12 hours, the old culture medium was discarded, the cells were washed once with PBS, and 100 μL of 0, 50, 100, 150, 200, 250, and 300 μg mL LPS was added to each well. -1 Containing M-MPDA@CeO 2 After overnight culture, discard the old culture medium, add 100 μL of serum-free culture medium containing 10% CCK-8 to each well, and incubate at 37°C for 1 hour. Finally, measure the absorbance value at a wavelength of 450 nm to calculate the cell viability.

[0139] Cell transmission electron microscopy

[0140] (1) Sample preparation: MLE-12 cells were cultured at 5×10 5 The cells were planted in a 60 mm culture dish at a density of 100 cells per well. The control group was not treated. The model group was induced with complete medium containing 1 μg / ml LPS and then divided into 5 groups, namely control group, LPS, LPS+MPDA, LPS+MPDA@CeO 2 , LPS+M-MPDA@CeO 2, The cells were intervened and treated for 12 hours. After the cells were digested with trypsin, they were centrifuged at a speed of 1000 rpm for 5 minutes, and the cells were deposited in the centrifuge tube. After removing the supernatant, 3% glutaraldehyde fixative was slowly added along the tube wall using a pipette to resuspend the cells, and the cells were allowed to stand for 5 minutes in an environment at 4°C. Subsequently, the fixed cell suspension was transferred to a 1.5 mL conical bottom EP tube and centrifuged at a speed of 12000 rpm for 10 minutes. The supernatant was removed, and the cell pellet was retained. (2) Sample fixation: 3% glutaraldehyde was used for primary fixation to ensure complete infiltration of the entire sample. Subsequently, for the second round of fixation, 1% osmium tetroxide was used to enhance the stability of the sample. (3) Dehydration treatment: The cells underwent a stepwise dehydration process using acetone with concentrations gradually changing to 30% → 50% → 70% → 80% → 90% → 95% → 100% (the 100% concentration was changed 3 times) to gradually remove the moisture in the sample. (4) Infiltration and embedding process: Using dehydrating agent and Epon812 embedding agent, gradual infiltration steps were carried out in the ratios of 3:1, 1:1, and 1:3. Finally, Epon812 embedding was performed to ensure that the sample was completely embedded in the resin. (5) Preparation of ultra-thin sections: Using an ultra-microtome, ultra-thin sections of approximately 60 - 90 nm were prepared to ensure that the thickness of the sections met the requirements of the electron microscope. The ultra-thin sections were unfolded and transferred onto copper grids in preparation for subsequent staining and observation. (6) Staining process: First, uranyl acetate was used for staining for 10 - 15 minutes to improve the contrast of cell organelles. Subsequently, lead citrate was used for staining for 1 - 2 minutes, both at room temperature. (7) Electron microscope observation: The samples on the copper grids were imaged using a JEM-1400FLASH transmission electron microscope. Each copper grid was observed at 8000 times magnification to collect images.

[0141] Live and dead cell staining

[0142] MLE-12 cells with good growth status were seeded in a 12-well plate at a cell density of 2×10 4 cells / well and cultured overnight for adhesion. The control group was not treated. After the model group was induced with complete medium containing 1 μg / ml LPS, the original medium was removed and the cells were gently washed once with PBS. A total of 5 groups were divided, namely the control group, LPS, LPS + MPDA, LPS + MPDA@CeO 2 , LPS + M-MPDA@CeO 2 . After the cells were intervened and treated for 12 hours according to the above grouping, the original medium was removed and the cells were washed. The live and dead cell staining solution (0.5% calcein and 2% PI staining solution) was prepared according to the instructions. 500 μL of the staining solution was taken for each well to cover the cells completely. After incubating in an incubator at 37°C for 30 minutes, the cells were washed 2 - 3 times with PBS. Another 500 μL of fresh medium was added to each well, and the survival status of the cells in each group was observed under a fluorescence microscope.

[0143] Mitochondrial membrane potential

[0144] Mitochondrial membrane potential staining was used to observe the changes in mitochondrial membrane potential of cells in each group after being treated with different methods. MLE-12 cells (2×10 4 cells / dish) were cultured in a 12-well plate. After cell modeling, the cells were intervened and treated according to the experimental grouping. Then, the JC-1 solution prepared according to the instructions was added to the above culture dishes. After continuing to incubate in an incubator at 37 °C for 20 min, the culture dishes were washed with JC-1 buffer, and fluorescence microscopy imaging was performed.

[0145] Uptake of nano-complexes by MLE-12 cells

[0146] Fluorescence

[0147] To study the phagocytosis efficiency of MLE-12 cells on nanoparticles, M-MPDA@Cy5.5 nano-complexes loaded with the fluorescent dye Cy5.5 were co-cultured with MLE-12 cells for different times (0 h, 0.5 h, 1 h, 2 h, 3 h). The cell nuclei were stained with DAPI dye, and the red fluorescence distribution of Cy5.5 in the cells was observed by fluorescence microscopy.

[0148] ICP-OES

[0149] Cells were seeded in a 6-well plate at a density of 7×10 5 cells per well. After culturing overnight, the original culture medium was removed, and 1.5 ml of fresh medium containing M-MPDA@CeO 2 (200 μg / ml) was added to each well. After continuing to culture for different times, cell precipitates were collected in centrifuge tubes at 0.5 h, 2 h, 4 h, 8 h, 12 h, and 24 h respectively. Then, they were digested overnight with aqua regia to a final volume of 5 mL, and the uptake amount of Ce by MLE-12 cells at each time point was measured by ICP-OES (n = 3).

[0150] Detection of ROS content in MLE-12 cells

[0151] Cells were seeded in a 24-well plate at a density of 5×10 4 cells per well and divided into 5 groups, namely the control group, LPS, LPS+MPDA, LPS+MPDA@CeO 2 , LPS+M-MPDA@CeO 2, After culturing cells in groups for 12 hours, remove the medium, wash twice with PBS, add 500 μl of DCFH-DA reactive oxygen species probe diluted with basal medium (dilution ratio is 1:1000) to each well, continue to incubate in the dark for 20 min, and shake once every 5 min. After incubation, discard the medium, wash the cells twice with PBS, and then observe under an inverted fluorescence microscope or quantitatively analyze the fluorescence intensity by flow cytometry (excitation = 480 nm, emission = 525 nm).

[0152] M-MPDA@CeO 2 Effect on gene expression levels of RAW264.7 cells

[0153] Obtaining oxidative stress genes

[0154] Visit the official website of GeneCards (http: / / www.genecards.org / ), search for oxidative stress-related keywords: "oxidative stress", "ROS", "antioxidant", and intercept genes with a relevance score greater than 10. Take the intersection of the obtained oxidative stress-related genes and the differentially expressed genes obtained by sequencing.

[0155] Protein-protein interaction network analysis

[0156] The protein-protein interaction (PPI) network involves physical or functional contacts between two or more proteins and is used to reveal the functional connections and biological processes between intracellular proteins. Use the String database (http: / / string-db.org) for protein-protein interaction network analysis. Input the intersection targets to construct a protein-protein interaction network, import it into the Cytoscape 3.9.1 software to construct an interaction relationship network diagram, and apply the CytoHubba plugin to screen and rank key genes in the biological network.

[0157] GO enrichment analysis

[0158] Gene Ontology (GO) is an important tool for describing the functions of genes and proteins, which mainly covers three aspects: Biological Process (BP), Cellular Component (CC), and Molecular Function (MF). The intersection genes obtained in the previous step are associated with each term in the GO database, and the number of genes in each term is calculated. Using the hypergeometric test, GO terms significantly enriched in the differential genes are analyzed to further understand the functional characteristics of the intersection genes in terms of biological processes, cellular components, and molecular functions compared with the whole genome background.

[0159] KEGG enrichment analysis

[0160] KEGG enrichment analysis is used to identify significantly enriched terms related to KEGG pathways in the differentially expressed gene set. By mapping the differentially expressed genes to the KEGG pathways and counting the number of genes in each pathway, the enrichment analysis compares the differences between the differentially expressed gene set and the whole genome background, thereby identifying whether there is significant enrichment in specific pathways.

[0161] The CCK-8 method was used to study the effect of M-MPDA@CeO 2 on cell viability. As Figure 11 shown, after treating cells with different concentrations of M-MPDA@CeO 2 , M-MPDA@CeO 2 showed no significant toxicity to both cell lines at a concentration of 200 μg / mL, and the cell viability remained at about 90%. Therefore, 200 μg / mL was selected as the working concentration of M-MPDA@CeO 2 in this experiment.

[0162] M-MPDA@CeO 2 protects MLE-12 cells from oxidative stress damage

[0163] As Figure 12 shown, the cell viability decreased significantly after LPS treatment and gradually recovered with the increase in the treatment concentration of M-MPDA@CeO 2 , indicating that M-MPDA@CeO 2 has a protective effect on LPS-induced oxidative damage of cells.

[0164] Transmission electron microscopy was used to observe the morphological and structural changes of mitochondria in LPS-induced MLE-12 cells after different intervention treatments ( Figure 13)。The morphological structure of mitochondria in the control group cells was normal. The shape of mitochondria in the cytoplasm was nearly oval, with clear and straight cristae, uniform electron density of the matrix, and it was gray. After LPS induction, the morphological structure of cell mitochondria showed obvious abnormalities. A large number of mitochondria in the cytoplasm were significantly shrunk, with smaller volume, showing a thin rod shape or spherical shape, the cristae were reduced or broken, the inter-cristal space was significantly widened or even ruptured, and the electron density of the matrix was deepened. After treatment with MPDA, MPDA@CeO 2 , M-MPDA@CeO 2 , the degree of mitochondrial damage gradually decreased compared with the model group. Especially in the M-MPDA@CeO 2 group, only mild swelling of mitochondria was observed after treatment, which was similar to the normal mitochondrial structure.

[0165] Calcein / PI was used to label live cells and dead cells. As shown in Figure 14 A-B, compared with the control group, the number of live cells in the model group decreased significantly. After treatment with MPDA, MPDA@CeO 2 , M-MPDA@CeO 2 respectively, the number of dead cells in MLE-12 cells gradually decreased, and the number of live cells gradually increased.

[0166] Mitochondrial membrane potential is the potential difference across the mitochondrial membrane in cells. It is one of the key factors maintaining mitochondrial function and energy production, and plays an important role in cell metabolism, survival and apoptosis processes. The changes in mitochondrial membrane potential of cells after different treatment methods are shown in Figure 15 . Compared with the control group, the red fluorescence signal in the model group almost disappeared. After the model group cells were treated with MPDA, MPDA@CeO 2 , M-MPDA@CeO 2 respectively, it was observed that the red fluorescence signal gradually increased and the green fluorescence signal gradually decreased, indicating that after treatment with nanomaterials, the damaged mitochondria could be repaired to a certain extent. Most strikingly, in the model group + M-MPDA@CeO 2 treatment group, we observed a significant increase in the red fluorescence signal and a decrease in the green fluorescence signal, indicating that M-MPDA@CeO 2 could well repair the mitochondrial damage of MLE-12 cells after LPS stimulation and protect cells from apoptosis caused by oxidative stress.

[0167] As a potent extracellular stimulant, LPS can induce oxidative stress in MLE-12 cells, leading to damage to cell structure and function. In this study, the viability of MLE-12 cells after LPS treatment decreased significantly, and mitochondrial morphology was abnormal, including reduced volume, broken cristae, and increased electron density, which are direct evidence of cell damage. As the energy factory of cells, the structural and functional integrity of mitochondria is essential to maintain cell survival. The decrease in mitochondrial membrane potential usually reflects the damage of mitochondrial function, which further leads to energy metabolism disorders and cell death. The above experimental results show that MPDA has a certain antioxidant effect, and MPDA@CeO 2 It has synergistic antioxidant function. M-MPDA@CeO 2 MPDA@CeO 2 After modification of the macrophage membrane, the surface phospholipid bilayer structure increases the cell's uptake of nanomaterials, and its antioxidant function is positively correlated with the quality of the material.

[0168] Uptake of nanocomplexes by MLE-12 cells

[0169] In order to verify the uptake function of nanocomplexes by MLE-12 cells under in vitro conditions, MLE-12 cells were co-incubated with M-MPDA@Cy5.5. By fluorescence microscopy at different time points, we found that significant red fluorescence signals were gathered around the nuclei of MLE-12 cells treated with M-MPDA@Cy5.5, and the intensity of the fluorescence signal increased with time ( Figure 16A -B), indicating that the uptake of nanocomplexes by MLE-12 cells was positively correlated with time.

[0170] ICP-OES analysis of MLE-12 cell uptake of M-MPDA@CeO 2 The Ce content in the single-well cell was then quantitatively detected. Figure 16C As shown in the figure, with the extension of incubation time, the Ce content in the single-well cells gradually increased. When incubated for 8 hours, the total Ce content in the single-well cells was about 63.09±8.42ng / Well, and then with the extension of incubation time, the Ce content did not increase significantly, suggesting that MLE-12 cells may be sensitive to M-MPDA@CeO 2 The intake has reached saturation.

[0171] In order to evaluate the M-MPDA@CeO 2 The ability to scavenge ROS at the cellular level was verified by fluorescence microscopy imaging using a classic ROS probe (DCFH-DA). Figure 17A , Figure 17BAs shown, compared with the control group, the significantly enhanced green fluorescence signal in the model group cells indicated that excessive reactive oxygen species were generated in the cells after LPS stimulation. After the cells in the model group were treated with MPDA, MPDA@CeO 2 , M-MPDA@CeO 2 respectively, the fluorescence signal gradually weakened. Compared with the MPDA@CeO 2 group, the ROS fluorescence signal in the cells treated with M-MPDA@CeO 2 was weaker, and the difference was statistically significant with P < 0.05. The results of flow cytometry detection of ROS were consistent with the fluorescence results ( Figure 17C ).

[0172] After RAW264.7 cells were treated according to different experimental groups for 24 h, qRT-PCR was used to detect the pro-inflammatory related genes TNF-α, IL-1β, IL-6, as well as the M1 macrophage markers iNOS, CD86 and the M2 macrophage markers Arg1, CD206. The results were as Figure 18 shown. Compared with the control group, the expressions of the inflammatory genes TNF-α, IL-1β, and IL-6 in the model group were all up-regulated (P < 0.001). The gene expressions of the M1 macrophage markers iNOS and CD86 were also up-regulated. After treatment with MPDA, MPDA@CeO 2 , M-MPDA@CeO 2 respectively, compared with the model group, the expression levels of inflammatory factor genes in each group were down-regulated to varying degrees (P < 0.05). Compared with the MPDA@CeO 2 group, the down-regulation of the expression levels of inflammatory factor genes in the M-MPDA@CeO 2 group was more obvious (P < 0.05), and the difference was statistically significant. The expression levels of the M2 macrophage-related genes Arg1 and CD206 were up-regulated compared with the model group after intervention with M-MPDA@CeO 2 (P < 0.05). The experimental results showed that after LPS stimulation, the secretion of pro-inflammatory factors by RAW264.7 cells increased and polarized towards M1 macrophages. M-MPDA@CeO 2 could inhibit the expression of macrophage inflammatory factors and regulate the conversion of M1 macrophages to M2 macrophages.

[0173] The polarization of macrophages was detected by flow cytometry to detect the expressions of CD86 and CD206. The results were as Figure 19 shown. In the model group, the proportion of CD86-positive cells was significantly higher than that in other groups, indicating that LPS stimulation up-regulated the expression of CD86. After treatment with MPDA, MPDA@CeO 2 and M-MPDA@CeO 2After different treatments, the proportion of CD86-positive cells decreased, while the proportion of CD206-positive cells gradually increased. The results showed that both MPDA and MPDA@CeO 2 had the ability to inhibit the expression of CD86 and increase the expression of CD206 to a certain extent. The M-MPDA@CeO 2 nano-complex synergistically enhanced the ability to inhibit the expression of CD86 and increase the expression of CD206. The experimental results showed that M-MPDA@CeO 2 could inhibit the polarization of M1 macrophages and promote the polarization of M2 macrophages.

[0174] Using molecular biology techniques and related chemical reagents, the 6 RNA samples of this project were subjected to tissue fragmentation, nucleic acid extraction and purification, and then detected and quality controlled, in order to obtain total RNA samples that meet the requirements for subsequent library construction. Clear bands were detected in each RNA sample, and no obvious degradation was seen, indicating that the RNA quality of each sample was qualified for subsequent experiments.

[0175] A total of 20,263 genes were detected in this sequencing. We screened these genes under the conditions of P < 0.05 and log2FoldChange ≥ 0.5, and obtained a total of 1,343 significantly differentially expressed genes.

[0176] Oxidative stress genes were obtained from genecard. A total of 646 oxidative stress-related genes were obtained by intercepting genes with a correlation score greater than 10. The oxidative stress-related genes were intersected with the differentially expressed genes obtained from the above sequencing (OS-DEGs), and a total of 50 intersection genes were obtained.

[0177] The protein-protein interaction network had 43 nodes and 165 interaction relationships. Using the MCC algorithm in the Cytohubba plugin of Cytoscape software, 10 key genes were screened, which were: FOS, NFKBIA, EGFR, CEBPB, RELA, FOXO1, EGR1, CREBBP, CYCS, HSPA5.

[0178] The GO enrichment analysis was performed on the OS-DEGs intersection genes, and 54 GO terms were obtained by screening according to P < 0.05. The biological process functions (BP) of the OS-DEGs intersection genes were mainly enriched in responses to hypoxia, inflammatory responses, fatty acid β-oxidation, positive regulation of NIK / NF-kappaB signaling, cellular responses to hypoxia, etc.

[0179] KEGG enrichment analysis was performed on the intersection genes of OS-DEGs using the R package, and screening was carried out according to P < 0.05. A total of 106 signaling pathways or biochemical pathways were obtained. The pathways closely related to oxidative damage were mainly enriched in the HIF-1 signaling pathway, TNF signaling pathway, AMPK signaling pathway, IL-17 signaling pathway, oxidative phosphorylation, MAPK signaling pathway, PI3K-Akt signaling pathway, NF-kappa B signaling pathway, FoxO signaling pathway, etc.

[0180] To detect whether M-MPDA@CeO 2 would cause hemolytic reactions, whole blood from healthy mice was collected in anticoagulant tubes, mixed well and centrifuged at 1000 rpm / min for 5 min. The lower layer of red blood cells was taken and washed 3 times with PBS. Finally, the red blood cells were resuspended in PBS solution to make a 2% red blood cell suspension. 500 μl of M-MPDA@CeO 2 solutions at different concentrations (50 μg / ml, 100 μg / ml, 150 μg / ml, 200 μg / ml, 250 μg / ml, 300 μg / ml) were added to an equal volume of 2% red blood cell suspension. PBS was set as the negative control, and ddH 2 O was the positive control group. After thorough mixing, it was incubated in a water bath at 37 °C for 1 hour, and then centrifuged at 3000 rpm / min for 15 min. The samples were lined up and photographed. 100 μl of the supernatant in different test tubes was taken with a pipette and placed in a 96-well plate, and the absorbance values of different samples at 542 nm were measured with an enzyme-linked immunosorbent assay reader (n = 3). The hemolysis rate was calculated as follows:

[0181]

[0182] To evaluate the in vivo safety of M-MPDA@CeO 2 , the blood routine and serum biochemical indexes of mice were detected 2 weeks after administration. 10 healthy BALB / c mice were selected and randomly divided into 2 groups, with 5 mice in each group. One group was injected with M-MPDA@CeO 2 via the tail vein at a dosage of 10 mg / kg, corresponding to a Ce dosage of 0.634 mg / kg. Another group was injected with an equal volume of PBS as a control. The drug was administered once a day for 14 consecutive days, and the body weight of the mice was recorded every 2 days. On the 14th day, blood samples of each group of mice were collected by eye bleeding, and the white blood cell (WBC) count, red blood cell (RBC) count, hemoglobin (HGB), and platelet count (PLT) in the blood of each group were measured with a hematology analyzer. Serum biochemical indexes such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and serum creatinine (CREA) were detected with a serum biochemical analyzer. The measurement results were statistically analyzed and compared with the control group to evaluate M-MPDA@CeO 2For in vivo safety.

[0183] Construction of ALI mouse model

[0184] After healthy BALB / c mice were anesthetized by intraperitoneal injection of sodium pentobarbital, they were fixed in a supine position at a 45° slope. The tongue of the mouse was pulled out with forceps to expose the larynx. After tracheal intubation, lipopolysaccharide (dissolved LPS in sterile saline, 10 mg / kg) was instilled into the trachea using a fine syringe to construct an ALI mouse model. The control group of mice was only instilled with an equal volume of saline, and after instillation, they were placed in a cage to wake up naturally.

[0185] A small animal in vivo imaging system was used to study the in vivo targeting of the nanocomplex. 200 μl (200 μg / ml) of M-MPDA@Cy5.5 and MPDA@Cy5.5 were injected into ALI mice via the tail vein, respectively. After satisfactory anesthesia by intraperitoneal injection of sodium pentobarbital, the fluorescence signals in the mice were recorded under a small animal in vivo imaging system at 0.5, 2, 4, 6, 8, 12, and 24 h after injection of the nanomaterials. The mice were sacrificed 24 hours later, and the heart, liver, spleen, lung, and kidney tissues were taken to record the fluorescence signals.

[0186] Three 6-week-old healthy BALB / c mice were injected with M-MPDA@CeO via the tail vein at a dose of 5 mg / kg. 2 At 0, 0.5, 1, 2, 4, 6, 12, 24, 36, 48, and 72 h after injection, 20 μL of blood samples were taken from the medial canthus of the eye. The samples were digested overnight after being fixed to 5 mL with aqua regia (concentrated HNO 3 / concentrated HCl, v / v = 1:3), and the concentration of Ce in the blood samples at each time point was measured by inductively coupled plasma optical emission spectrometry (ICP-OES) (n = 3) to evaluate the circulation of M-MPDA@CeO 2 in the blood.

[0187] Evaluation of the therapeutic effect of acute lung injury

[0188] Twenty-five male BALB / c mice were evenly divided into 5 groups (n = 5): Control group, LPS group, LPS + MPDA group, LPS + MPDA@CeO 2 group, LPS + M-MPDA@CeO 2 group. The Control group was normal mice without modeling. The LPS + MPDA group, LPS + MPDA@CeO 2 group, and LPS + M-MPDA@CeO 2 group were administered by tail vein injection (the dosage was 200 μL 5 mg / kg) 2 hours after modeling. The Control group and the LPS group were injected with an equal volume of PBS via the tail vein.

[0189] Obtaining and analysis of bronchoalveolar lavage fluid samples

[0190] After anesthetizing the mice by intraperitoneal injection of sodium pentobarbital, the trachea was incised, and 0.8 mL of pre-cooled sterile PBS was slowly injected through the tracheal cannula, and lavage was performed three times to ensure that the withdrawal rate was greater than 60% each time. The collected BALF samples were aliquoted and placed in cryotubes, and immediately frozen at -80 °C in a refrigerator for subsequent detection. The total cell count in BALF was detected using a cell counter, the total protein concentration was measured using a BCA quantitative detection kit, and the inflammatory factors TNF-α, IL-1β, and IL-6 were detected using the corresponding ELISA kits.

[0191] Mouse lung wet / dry weight ratio

[0192] After collecting the intact lung tissue, the surface blood was gently aspirated using filter paper, and its wet weight (W) was recorded. Next, the lung tissue was dried in an oven at 70 °C for 72 hours until the weight no longer changed, and then its dry weight (D) was measured. Finally, the wet / dry weight ratio of the mouse lung tissue was calculated.

[0193] Survival curve

[0194] First, 40 male BALB / c mice were taken to construct an ALI mouse model with an LPS dose of 50 mg / Kg (lethal dose). After successful modeling, the mice were randomly divided into 2 groups, with 20 mice in each group, namely the LPS group and the LPS+M-MPDA@CeO 2 group. In the LPS+M-MPDA@CeO 2 group, 2 hours after modeling, M-MPDA@CeO was injected via the tail vein 2 (the dosage was 200 μL), and the LPS group was injected with an equal volume of PBS via the tail vein. The survival days of each mouse were recorded, with 1 representing death and 0 representing survival.

[0195] Pathological detection of lung tissue

[0196] (1) HE staining: The mice were sacrificed after 24 hours, and then the lung tissue (lower right lobe) was removed and fixed with 4% paraformaldehyde. The fixed lung tissue was embedded in an appropriate tissue embedding agent and prepared into sections. The sections were placed in a ventilated place to dry. Subsequently, the sections were subjected to routine HE (hematoxylin and eosin) staining to clearly show the cell nucleus and cytoplasmic structures in the tissue, and the pathological section characteristics of the mouse lung tissue were observed in detail under a microscope.

[0197] (2) Tunel staining: First, fix, embed, section, and dewax the collected lung tissue samples. Subsequently, perform protease digestion pretreatment to enhance the permeability of the staining solution. Then, use a TUNEL staining kit and follow the detailed operation manual provided by the manufacturer for the staining steps, including defatting, repair, TUNEL reaction, washing, and nuclear staining of the sections. Then observe the Tunel-positive cells under a fluorescence microscope.

[0198] (3) MDA immunohistochemistry: First, dewax the paraffin sections to water treatment, and sequentially place them in an environmentally friendly dewaxing solution and absolute ethanol for defatting to ensure the integrity of the tissue sections. Then perform antigen repair. Subsequently, block endogenous peroxidase by incubating the sections in a 3% hydrogen peroxide solution and then wash with PBS. Then perform serum blocking by covering the tissue with 3% BSA for blocking treatment. Next, perform primary and secondary antibody treatments by dropping the corresponding antibodies on the sections and incubating overnight. Then perform DAB color development, observe the color development under a microscope, and the positive is brownish yellow. Finally, perform nuclear counterstaining and dehydration and mounting treatments, and check and interpret the results under a white light microscope.

[0199] (4) Tissue reactive oxygen species staining: First, warm the frozen sections at room temperature and drain the water, then draw a circle around the tissue with a histochemical pen and add a spontaneous fluorescence quencher to eliminate the interference of background spontaneous fluorescence. Subsequently, perform ROS staining by adding the ROS staining solution into the circle and incubating in an incubator to label the ROS distribution in the tissue. Then perform DAPI staining to counterstain the nucleus. Finally, perform mounting treatment and collect images under a fluorescence microscope.

[0200] To investigate the hemolytic toxicity of the M-MPDA@CeO 2 nano-composite, a hemolysis experiment was carried out by co-incubating red blood cells with the nanomaterial in vitro. Among them, ddH 2 O was used as a positive control, and PBS was used as a negative control. The results are as Figure 19 shown. When the concentration of M-MPDA@CeO 2 reached 300 μg / ml, the calculated hemolysis rate was only (2.09 ± 0.24)%.

[0201] To evaluate whether there is potential systemic toxicity during the in vivo application of M-MPDA@CeO 2 , this study continuously detected the blood routine parameters, serum biochemical indexes, and body weight changes of mice after 2 weeks of drug administration. The results of the blood routine test showed that all parameters detected by a blood cell counter after 2 weeks of M-MPDA@CeO 2 drug administration were within the normal range and there was no significant difference compared with the control group; the results of the serum biochemical index test showed that M-MPDA@CeO 2There was also no significant difference between the administration group and the control group ( Figure 20 ); compared with the control group, there were no significant abnormalities in the main organs such as the heart, liver, spleen, lungs, and kidneys of the mice in the administration group ( Figure 21 ); the results of body weight monitoring showed that M-MPDA@CeO 2 there was no significant difference in the body weight change of the mice in the administration group compared with the control group during the entire administration process ( Figure 22 ). These results indicate that M-MPDA@CeO 2 has no obvious toxic and side effects at the current administration dose and has good biocompatibility in vivo.

[0202] The nanomaterials were injected into the ALI mice via the tail vein, and the fluorescence distribution was observed at different time points. As shown in Figure 23(A), relatively clear fluorescence signals began to appear in the lungs 4 hours after the injection of M-MPDA@Cy5.5, and then the fluorescence signals mainly aggregated in the lungs. However, no obvious fluorescence signal enrichment was observed in the lungs after the injection of MPDA@Cy5.5 without macrophage cell membrane coating. The mice were sacrificed 24 hours after the injection of the materials to obtain the main organs for fluorescence imaging ( Figure 23B 、 Figure 23C ). Strong fluorescence signals were detected in the liver and kidney tissues of both groups of mice. It is speculated that the nanocomplex is mainly metabolized by the liver and kidneys; compared with the MPDA@Cy5.5 group, the fluorescence intensity in the lung tissue of the M-MPDA@Cy5.5 group was stronger, indicating that the nanocomplex wrapped with macrophage cell membrane can effectively accumulate in the pulmonary inflammatory injury site. This experiment further revealed the dynamic process of the distribution and accumulation of the nanocomplex in vivo, verifying that the macrophage cell membrane biomimetic nanomaterial can simulate the surface characteristics and biological activities of macrophages. Through this biomimetic nanomaterial, precise targeting of the lung injury site can be achieved.

[0203] To reveal the circulation law of M-MPDA@CeO 2 in the blood, ICP-OES was used to quantitatively analyze the cerium element concentration in the blood of mice at different time points after the injection of the nanomaterials. The results are as Figure 24 shown. The blood half-life of M-MPDA@CeO 2 was 10.53 ± 1.61 h, which may be attributed to the effective avoidance of the rapid phagocytosis of nanoparticles by macrophages due to the coating of the macrophage cell membrane, thus prolonging its circulation time. This escape mechanism helps M-MPDA@CeO 2 maintain a longer blood half-life in vivo, thereby improving its stability and pharmacokinetic performance in vivo.

[0204] Total number of cells in BALF

[0205] The total cell count in bronchoalveolar lavage fluid (BALF) is commonly used to evaluate the degree of lung inflammation. Since the inflammatory response causes white blood cells (especially macrophages and neutrophils) to enter the alveoli from the blood, the total cell count in BALF increases. Therefore, the determination of the total cell count in BALF is of great significance for diagnosing and monitoring inflammatory activities in lung diseases. The total cell count in BALF of mice in each group was measured using a cell counter, and the results are as Figure 25 shown. The cell count in the LPS model group was significantly higher than that in the control group (P < 0.001), reaching (298.89 ± 18.6) × 10 4 , demonstrating the successful establishment of the ALI mouse model. Compared with the model group, the total cell count in BALF of each treatment group decreased to varying degrees. Among them, the total cell count in BALF of the LPS + MPDA@CeO 2 and LPS + M-MPDA@CeO 2 treatment groups decreased to (111.77 ± 20.19) × 10 4 and (49.43 ± 14.95) × 10 4 respectively.

[0206] Total protein concentration in BALF

[0207] In the evaluation of lung injury, the protein concentration in bronchoalveolar lavage fluid is regarded as a key indicator to measure the change in the permeability of pulmonary capillaries. This is because when the lung is damaged, the permeability of capillaries increases, resulting in more proteins leaking from the blood vessels into the alveoli. Therefore, the increase in the protein concentration in BALF directly reflects the degree of lung injury and the permeability state of capillaries. The total protein concentration in BALF of ALI mice in each treatment group was measured using a BCA protein quantification kit, and the results are as Figure 26 shown. Compared with the control group, the LPS model group had a significant increase, reaching (647.61 ± 23.26) μg / mL, indicating that the permeability of alveolar epithelial microvessels increased after LPS stimulation, leading to a large amount of protein leakage. After intervention with nanomaterials, the protein concentration in BALF of each treatment group decreased to varying degrees. Among them, the protein concentration in BALF of the LPS + MPDA@CeO 2 and LPS + M-MPDA@CeO 2 treatment groups decreased to (437.27 ± 46.55) and (238.53 ± 45.34) μg / mL respectively.

[0208] Wet / dry ratio of lung tissue

[0209] The wet / dry weight ratio of the lung, as a direct method for measuring the degree of pulmonary edema, is also an evaluation index highly sensitive to lung tissue injury. The increase in edema means abnormal accumulation of water in lung tissue, which is usually closely related to the severity of lung tissue injury. Therefore, the wet / dry weight ratio of the lung not only provides an intuitive measurement of the level of pulmonary edema but also indirectly reveals the scope and degree of lung tissue injury. Twenty-four hours after modeling, the wet / dry weight of the right lung of mice in each treatment group was measured. The results are shown in Figure 27 . Compared with the control group, the wet / dry weight ratio of mice in the model group increased significantly (P < 0.001), indicating a significant increase in pulmonary edema. When compared with the model group, the wet / dry weight ratio of the MPDA treatment group did not show a statistically significant change, while that of the MPDA@CeO 2 treatment group decreased slightly (P < 0.05). Most notably, the wet / dry weight ratio of the M-MPDA@CeO 2 treatment group decreased significantly, indicating that M-MPDA@CeO 2 showed the best effect in inhibiting pulmonary edema.

[0210] During the pathogenesis of ALI, numerous inflammation-related cells are activated, such as neutrophils and macrophages. These cells release a large number of inflammation-promoting factors, exacerbating lung tissue injury. The contents of TNF-α, IL-1β, and IL-6 in the BALF supernatant were measured using an ELISA kit. The results are shown in Figure 28. Compared with the control group, the concentrations of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 in the LPS-induced model group increased significantly, indicating an out-of-control inflammatory response in vivo. After different grouping interventions, each group showed a certain effect of reducing inflammatory factors. The concentration of inflammatory factors in the M-MPDA@CeO 2 group decreased significantly, indicating that this treatment strategy showed excellent effects in significantly inhibiting the inflammatory response.

[0211] An ALI mouse model was constructed using a lethal dose of LPS, and the survival times of mice in the M-MPDA@CeO 2 intervention group and the control group were recorded. As shown in Figure 29 , on the 8th day, all 20 control group mice died, while when recorded until the 14th day, more than half of the mice in the M-MPDA@CeO 2 intervention group were still alive.

[0212] Histopathological evaluation of lung tissue

[0213] (1) The results of H&E staining are shown in Figure 30As shown, there were a large number of inflammatory cell infiltrations in the lung tissues of the LPS-induced model group, the alveolar structure was damaged, and a large number of red blood cells infiltrated, resulting in an enlarged alveolar wall space. After treatment with different groups, both the MPDA and MPDA@CeO 2 treatment groups showed varying degrees of improvement. And in the M-MPDA@CeO 2 treatment group, the improvement effect was the most obvious. This treatment group could significantly reduce the thickness of the alveolar septum, alleviate the degree of inflammatory cell infiltration, and effectively inhibit the occurrence of pulmonary interstitial and alveolar edema.

[0214] (2) The results of Tunel staining were as Figure 31 shown. Scattered green fluorescent signals were seen in the lung tissues of the LPS-induced model group, indicating apoptosis of tissue cells after LPS stimulation. Subsequently, the green fluorescent signals in the lung tissues of each treatment group gradually decreased, effectively demonstrating that the M-MPDA@CeO 2 nano complex could protect lung tissues from oxidative stress damage.

[0215] (3) MDA (malondialdehyde) is one of the important products of lipid peroxidation caused by reactive oxygen species. Lipid peroxidation is the result of intracellular oxidative stress. By measuring the content of MDA, the degree of lipid peroxidation can be understood, and then the degree of oxidative stress can be evaluated. The content of MDA in the lung tissues of mice in each group was detected by immunohistochemistry. The measurement results of MDA were as Figure 32 shown. In the LPS-induced model group, due to severe inflammation and lipid peroxidation reactions, the content of MDA increased significantly. After various treatment interventions, a downward trend in the content of MDA was observed. In particular, after treatment with M-MPDA@CeO 2 , the most significant effect of reducing the degree of inflammation and lipid peroxidation in vivo was shown. This indicates that M-MPDA@CeO 2 has excellent potential in alleviating oxidative stress and inflammatory reactions.

[0216] (4) The results of ROS frozen section staining were as Figure 33 shown. After LPS stimulation, the lung tissues of the model group showed red fluorescence throughout the field of view, indicating excessive accumulation of reactive oxygen species in the lung tissues due to acute lung injury. This was consistent with the previous experimental results. After treatment with MPDA and MPDA@CeO 2 , it was observed that these two materials showed the ability to synergistically scavenge reactive oxygen species. After treatment with M-MPDA@CeO 2 , the reactive oxygen species in the damaged lung tissues were significantly reduced. This further demonstrated that M-MPDA@CeO 2 has excellent reactive oxygen species scavenging ability in the in vivo environment.

[0217] In this study, by evaluating the hemolytic activity, physiological and biochemical indices, body weight changes, and pathological alterations of major organs of M-MPDA@CeO 2 , we demonstrated the high biocompatibility and safety of M-MPDA@CeO 2 at therapeutic doses.

[0218] Subsequently, the targeting ability of the nanocomplex to the sites of pulmonary inflammatory injury was verified by in vivo imaging of small animals. By injecting M-MPDA@Cy5.5 via the tail vein and tracking its distribution in mice using in vivo imaging technology for small animals, the results showed that the fluorescence signal could accumulate significantly in the lungs, indicating that the nanocomplex had good pulmonary targeting. Subsequently, we evaluated the therapeutic effect of M-MPDA@CeO 2 on the LPS-induced ALI mouse model. By comparing the pathological changes of lung tissues, levels of pulmonary inflammatory factors, survival curves, and ROS scavenging ability between the treatment group and the model group of mice, we found that M-MPDA@CeO 2 could significantly alleviate the lung injury caused by LPS, reduce the expression of inflammatory factors, and scavenge ROS.

[0219] The innovation of this study was mainly reflected in the design of a novel nanomaterial by applying the principle of bionics. The extracted macrophage membrane microcapsules were successfully coated on the surface of the mesoporous polydopamine-loaded cerium dioxide nanocomplex. By combining the excellent antioxidant functions of mesoporous polydopamine and cerium dioxide, and then applying the inflammatory tropism of macrophages, targeted therapy for acute lung injury was achieved.

Claims

1. A nanocomposite material, named M-MPDA@CeO2, characterized in that: The inner core is composed of mesoporous polydopamine loaded with ultra-small particle size cerium dioxide and is wrapped by macrophage membrane.

2. The method for preparing the nanocomposite material according to claim 1, characterized in that: The following steps are involved: (1) Preparation of the core: Add MPDA powder to anhydrous ethanol and stir, then add CeO2 nanoparticles after mixing, and stir at room temperature; centrifuge and wash, and vacuum freeze-dry to obtain MPDA@CeO2 powder; (2) Macrophage membrane encapsulation: The macrophage membrane microcapsules are fully mixed with the MPDA@CeO2 core aqueous solution, the mixed solution is ultrasonically treated, and then extruded back and forth under a 200 nm porous membrane to obtain an M-MPDA@CeO2 solution; the M-MPDA@CeO2 solution is vacuum freeze-dried to obtain the nanocomposite material M-MPDA@CeO2.

3. The method for preparing the nanocomposite material according to claim 2, characterized in that: The CeO2 nanoparticles in step (1) are prepared by the following method: Weigh 500 mg Ce(NO3)3·6H2O and 200 mg tri-n-octylphosphine oxide into a beaker, add 20 mL anhydrous ethanol and 2 ml deionized water, heat and stir to mix, when the solute is completely dissolved, add 5 ml octadecene and 100 μL oleylamine, continue to heat to 280 ° C under nitrogen protection for 1 hour; after cooling to room temperature, wash with anhydrous ethanol, centrifuge and discard the supernatant; then add the precipitate to acetone solution for solvent exchange, centrifuge again to remove the supernatant and open it to stand, and vacuum freeze-dry the precipitate to obtain a light yellow powder, which is CeO2 nanoparticles.

4. The method for preparing the nanocomposite material according to claim 2, characterized in that: The MPDA powder is prepared by the following method: 150 mg of dopamine hydrochloride and 100 mg of F127 were dissolved in 10 mL of deionized water and 10 mL of ethanol solution, respectively; after being fully dissolved, the above solutions were mixed, 160 μL of 1,3,5-trimethylbenzene was added to the mixed solution, and water bath sonication was performed; 375 μL of ammonia water was slowly added to the system under magnetic stirring conditions, and the mixed solution was reacted at 50° C. for 2 hours; after centrifugation, the precipitate was washed with ethanol and ultrapure water respectively; the final product was vacuum freeze-dried to obtain MPDA powder.

5. The method for preparing the nanocomposite material according to claim 2, characterized in that: In step (1), the ratio of MPDA powder to anhydrous ethanol is 20 mg: 20 mL; the amount of CeO2 added is 5 mg.

6. The method for preparing the nanocomposite material according to claim 2, characterized in that: The macrophage membrane microcapsules are prepared by the following method: (1) Mouse RAW 264.7 macrophage cell line was cultured in a 37°C, 5% CO2 constant temperature cell culture incubator using DMEM complete medium containing 10% serum; when the cell confluence rate reached 90%, the cells were gently washed three times with PBS; the cells were scraped and transferred to a centrifuge tube, centrifuged and the supernatant was discarded, and the cell pellet at the bottom of the tube was collected; (2) Cell membrane extraction: Add membrane protein extraction buffer to a centrifuge tube, pipette and mix well, and place on ice; The pretreated cell suspension was collected into a pre-cooled cell homogenizer and homogenized slowly for several times to fully lyse the cells; the product of the previous step was then collected into a centrifuge tube, centrifuged, and the supernatant was collected into another centrifuge tube; The obtained sediment at the bottom of the tube is the macrophage membrane fragments after centrifugation; the macrophage membrane fragments are resuspended in PBS and extruded multiple times through a 400 nm polycarbonate porous membrane using a liposome extruder to prepare macrophage membrane microcapsules.

7. The method for preparing the nanocomposite material according to claim 2, characterized in that: The ratio of mixing the macrophage membrane microcapsules and the MPDA@CeO2 core aqueous solution is 10:1 by mass.

8. The method for preparing the nanocomposite material according to claim 2, characterized in that: The conditions for ultrasonic treatment of the mixed solution are to use an ultrasonic disperser at a frequency of 30 kHz and a power of 100 W for 5 minutes.

9. Use of the nanocomposite material according to claim 1 in preparing a drug for treating ALI or ARDS caused by ALI.

Images