Engineered extracellular vesicle and application thereof

Abstract

According to the engineered extracellular vesicle and the application thereof provided by the invention, curcumin is loaded by adopting a co-incubation method, so that the loading capacity and the encapsulation efficiency of curcumin in unit extracellular vesicle are remarkably improved; the IgBD is adopted to modify the extracellular vesicles, so that the residence time of the extracellular vesicles in vivo is remarkably prolonged, the half-life period of the extracellular vesicles is prolonged, and the acute lung injury is improved and treated.

Description

Technical Field

[0001] This invention relates to the field of extracellular vesicle technology, specifically to an engineered extracellular vesicle and its applications. Background Technology

[0002] Extracellular vesicles (EVs) are vesicles released naturally from cells. Their main components include proteins, lipids, and nucleic acids, and they are characterized by high stability, high biocompatibility, and low immunogenicity and toxicity. EVs can load various molecules (such as miRNA, siRNA, DNA, proteins, and chemical drugs), and their unique membrane fusion mechanism enables them to achieve intercellular substance transfer without relying on specific receptors, making them a drug delivery carrier with enormous potential. However, the short circulation time of extracellular vesicles in vivo and the small dose delivered to the lesion site limit therapeutic efficacy.

[0003] Turmeric is a perennial herbaceous plant belonging to the ginger family (Zingiberaceae). Curcumin, extracted from turmeric, is a natural polyphenol with anti-inflammatory, antioxidant, antiviral, lipid-regulating, antithrombotic, antibacterial, immunomodulatory, hepatoprotective, anti-fibrotic, antidiabetic, antitumor, anticarcinogenic, immunomodulatory, and neuroprotective effects. It is used to treat common inflammatory diseases, tumors, and biliary tract diseases. Despite its numerous health benefits, curcumin's application remains limited and faces many challenges. For example, its low solubility in water is a key factor limiting its use. Improving curcumin's solubility is therefore of great importance.

[0004] Acute lung injury (ALI) is a severe respiratory disease prevalent worldwide, caused by both endogenous and exogenous pathogenic factors. It is primarily characterized by uncontrolled oxidative stress, alveolar epithelial damage, and inflammatory cell infiltration. Infectious pathogens, such as lipopolysaccharide (LPS), a major component of the cell wall of Gram-negative bacteria, have been shown to induce ALI. Once in the body, LPS is sensed by innate immune cells, triggering the secretion of inflammatory mediators and causing lung damage. Currently, treatments for ALI mainly include glucocorticoids, anti-inflammatory and antioxidant drugs, and mesenchymal stem cells (MSCs). Although current drugs can improve ALI symptoms, the prognosis is generally poor. The search for new drugs is urgently needed. Summary of the Invention

[0005] To address the aforementioned problems, a first aspect of the present invention provides an engineered extracellular vesicle modified with IgBD; the engineered extracellular vesicle is loaded with an active ingredient, namely curcumin.

[0006] As a preferred technical solution, the engineered extracellular vesicles have a curcumin loading of greater than 1.6 μg / 100 μg and a curcumin encapsulation rate of greater than 10%.

[0007] As a preferred technical solution, the protein content of the engineered extracellular vesicles is greater than 0.3 μg / μL.

[0008] IgBD is the immunoglobulin G binding domain, typically composed of 55-60 amino acids forming three antiparallel α-helices. When engineered extracellular vesicles are modified with IgBD, their clearance rate by the liver and spleen is significantly slowed, resulting in a substantial increase in their residence time (half-life) in the bloodstream.

[0009] A second aspect of the present invention provides a method for preparing engineered extracellular vesicles, comprising the following steps: Step S1, collect cell culture supernatant: after mesenchymal stem cells are cultured in complete culture medium, washed, and cultured in basal culture medium, collect the basal culture medium supernatant A; Step S2, Removal of cell debris: Cell debris is removed from supernatant A by centrifugation to obtain supernatant B; Step S3, preparation of extracellular vesicles: centrifuge the supernatant B and collect the precipitate C; wash and resuspend the precipitate C to obtain extracellular vesicle suspension D; Step S4, loading curcumin: Mix the extracellular vesicle suspension D with the curcumin solution evenly, and load the curcumin into the extracellular vesicles using a co-incubation method to obtain engineered extracellular vesicles.

[0010] As a preferred technical solution, the mesenchymal stem cells in step S1 are derived from mouse bone marrow; the complete culture medium is DMEM complete culture medium; the washing solution is PBS buffer; the cell density reaches more than 70% after culturing in the complete culture medium; the basal culture medium is serum-free DMEM basal culture medium; and the basal culture time is 24 hours.

[0011] As a preferred technical solution, the centrifugation speed in step S2 is 1200 rpm and the centrifugation time is 30 min.

[0012] As a preferred technical solution, in step S3, the centrifugal force is 120000×g, the centrifugation time is 2h, the centrifugation temperature is 4℃, and the washing and resuspending solution is PBS buffer.

[0013] As a preferred technical solution, in step S4, the solvent of the curcumin solution is DMSO; the concentration of curcumin in the curcumin solution is 1 mg / mL; the volume ratio of the exovesicle suspension D to the curcumin solution is 95:5; the concentration of curcumin during co-incubation is 50 μg / mL; the volume concentration of DMSO during co-incubation is 5%; the co-incubation temperature is 4℃; and the time is 24 hours.

[0014] As a preferred technical solution, the engineered extracellular vesicles are further modified with IgBD.

[0015] As a preferred technical solution, the IgBD modification method is as follows: IgBD-Cys is mixed with DSPE-PEG2000-MAL to prepare suspension E; the molar ratio of IgBD-Cys to DSPE-PEG2000-MAL is 1:7; a reducing agent is added to suspension E, and suspension F is obtained after incubation; the incubation temperature is 4℃, and the incubation time is 12 hours; the reducing agent is TCEP; the concentration of the reducing agent in suspension E is 5%. mmol / L; Dialysis suspension F, free DSPE-PEG2000-MAL was removed to obtain IgBD-DSPE; the molecular weight cutoff of the semipermeable membrane used in dialysis suspension F was 3KD; IgBD-DSPE was co-incubated with EV-CUR to obtain suspension G, dialysis suspension G, free protein was removed to obtain IgBD-modified EV-CUR, denoted as IgBD-EV-CUR; the co-incubation time was 12 hours; the molecular weight cutoff of the semipermeable membrane used in dialysis suspension G was 30KD.

[0016] The reducing agent is used to reduce the disulfide bonds in the protein, allowing the Cys side chain of the protein to fully react with MAL and form a stable covalent bond.

[0017] A third aspect of the present invention provides a pharmaceutical formulation comprising the aforementioned engineered extracellular vesicles.

[0018] As a preferred technical solution, the pharmaceutical preparation further includes cytokines, pH stabilizers, osmotic pressure regulators, and excipients. Cytokines include, for example, hepatocyte growth factor; osmotic pressure regulators include, for example, sodium chloride; and excipients include, for example, amino acids.

[0019] A fourth aspect of the present invention provides the use of the aforementioned engineered extracellular vesicles in a pharmaceutical formulation for the treatment of acute lung injury.

[0020] In a fifth aspect, the present invention provides a method for increasing the loading of curcumin in extracellular vesicles, the method employing the aforementioned engineered extracellular vesicle preparation method.

[0021] In a sixth aspect, the present invention provides a method for extending the half-life of extracellular vesicles, the method employing the aforementioned engineered extracellular vesicle preparation method.

[0022] A seventh aspect of the present invention provides the application of the aforementioned engineered extracellular vesicles in a drug delivery system.

[0023] Through the above technical solutions, the present invention achieves the following technical effects: (1) The co-incubation method was used to load curcumin, which significantly improved the loading capacity and encapsulation efficiency of curcumin in a unit of external vesicles.

[0024] (2) By modifying extracellular vesicles with IgBD, the residence time of extracellular vesicles in vivo is significantly increased and the half-life of extracellular vesicles is prolonged.

[0025] (3) Engineered extracellular vesicles EV-CUR and IgBD-EV-CUR can both improve acute lung injury. Attached Figure Description

[0026] Figure 1 The effect of EV-CUR in Example 1 on the cellular inflammation model is shown in the following rows: row A shows the expression results of inflammatory factors in A549 cells detected by quantitative real-time PCR; row B shows the expression results of inflammatory factors in BEAS-2B cells detected by quantitative real-time PCR; and row C shows the expression results of inflammatory factors in MLE-12 cells detected by quantitative real-time PCR. Figure 2 The effect of EV-CUR in Example 1 on a mouse model of acute lung injury and inflammation; Figure 2 A represents the total protein content in the bronchoalveolar lavage fluid of mice under different treatments; Figure 2 B represents the number of white blood cells in the bronchoalveolar lavage fluid of mice under different treatments; Figure 2 C represents the H&E staining of lung tissue from mice in each group; Figure 2 D represents the CD68 immunohistochemistry of lung tissue from mice in each group; Figure 2 E represents the inflammatory infiltration analysis of the lung tissue of mice in each group; Figure 2 F represents the statistical analysis of the proportion of CD68 in each group; Figure 3 Coomassie brilliant blue staining results for modifying the binding of proteins to extracellular vesicles; Figure 4 The results show the effect of IgBD modification on the half-life of extracellular vesicles in mice. Figure 5 The effect of IgBD modification on extracellular vesicle size; Figure 6 The effect of IgBD modification on extracellular vesicle morphology; Figure 7Fluorescence images showing the effect of IgBD modification on the aggregation of extracellular vesicles in the lungs; each row represents one replicate. Figure 8 Quantitative analysis of fluorescence intensity of extracellular vesicles in the lungs after IgBD modification; Figure 9 The effect of IgBD-modified extracellular vesicles on inflammation in MLE-12 cells; where A is a comparison of IL8 expression levels; B is a comparison of IL6 expression levels; C is a comparison of IL1β expression levels; and D is a comparison of TNFα expression levels. Figure 10 Statistics on total protein and white blood cell count in bronchoalveolar lavage fluid of mice under different treatments; among which Figure 10 The left side shows the total protein content in the bronchoalveolar lavage fluid of mice under different treatments; the right side shows the white blood cell count in the bronchoalveolar lavage fluid of mice under different treatments. Figure 11 The results of quantitative real-time PCR detection of inflammatory factors IL8, IL6, IL1β and TNFα in the lungs of mice under different treatments; Figure 12 H&E staining of lung tissues from mice under different treatments; Figure 13 Analysis of inflammatory infiltration in the lungs of mice under different treatments; Figure 14 CD68 immunohistochemistry of lung tissue from mice under different treatments; Figure 15 Statistical analysis of the proportion of CD68 immunohistochemical staining in lung tissues of mice under different treatments; In all the figures, **** indicates P < 0.0001, *** indicates P < 0.001, ** indicates P < 0.005, and * indicates P < 0.05; ns indicates no significant difference. Detailed Implementation

[0027] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the applicant provides an explanation and analysis through embodiments and comparative examples.

[0028] Example 1: Preparation of engineered extracellular vesicles (EV-CUR) by co-incubation method, including the following steps: Step S1: Collect cell culture supernatant: After mesenchymal stem cells are cultured in complete culture medium, washed, and cultured in basal culture medium, the basal culture supernatant A is collected; the mesenchymal stem cells are derived from mouse bone marrow; the complete culture medium is DMEM complete culture medium; the washing solution is PBS buffer; the cell density reaches more than 70% after culture in the complete culture medium; the basal culture medium is serum-free DMEM basal culture medium; the basal culture time is 24 hours. Step S2, Removing cell debris: Cell debris is removed from supernatant A by centrifugation to obtain supernatant B; the centrifugation speed is 1200 rpm and the centrifugation time is 30 min; Step S3, preparation of extracellular vesicles: centrifuge the supernatant B and collect the precipitate C; wash and resuspend the precipitate C to obtain extracellular vesicle suspension D, denoted as EV; the centrifugation force is 120000×g, the centrifugation time is 2 h, the centrifugation temperature is 4℃; the washing and resuspending solution is PBS buffer. Step S4, loading curcumin: Mix the extracellular vesicle suspension D with curcumin solution evenly, and load curcumin into the extracellular vesicles using a co-incubation method to obtain engineered extracellular vesicles, denoted as EV-CUR; In step S4, the solvent for the curcumin solution is DMSO; the concentration of curcumin in the curcumin solution is 1 mg / mL; the volume ratio of the external vesicle suspension D to the curcumin solution is 95:5; the concentration of curcumin during co-incubation is 50 μg / mL; the volume concentration of DMSO during co-incubation is 5%; and the co-incubation temperature is 4°C for 24 hours.

[0029] Example 2, preparation of engineered extracellular vesicles (IgBD-EV-CUR) by co-incubation method, including the following steps: Step S1: Collect cell culture supernatant: After mesenchymal stem cells are cultured in complete culture medium, washed, and cultured in basal culture medium, the basal culture supernatant A is collected; the mesenchymal stem cells are derived from mouse bone marrow; the complete culture medium is DMEM complete culture medium; the washing solution is PBS buffer; the cell density reaches more than 70% after culture in the complete culture medium; the basal culture medium is serum-free DMEM basal culture medium; the basal culture time is 24 hours. Step S2, Removing cell debris: Cell debris is removed from supernatant A by centrifugation to obtain supernatant B; the centrifugation speed is 1200 rpm and the centrifugation time is 30 min; Step S3, preparation of extracellular vesicles: centrifuge the supernatant B and collect the precipitate C; wash and resuspend the precipitate C to obtain extracellular vesicle suspension D; the centrifugation force is 120000×g, the centrifugation time is 2 h, the centrifugation temperature is 4℃; the washing and resuspending solution is PBS buffer. Step S4, loading curcumin: Mix the extracellular vesicle suspension D with the curcumin solution evenly, and load curcumin into the extracellular vesicles using a co-incubation method to obtain engineered extracellular vesicles loaded with curcumin, denoted as EV-CUR; The solvent for the curcumin solution is DMSO; the concentration of curcumin in the curcumin solution is 1 mg / mL; the volume ratio of the external vesicle suspension D to the curcumin solution is 95:5; the concentration of curcumin during co-incubation is 50 μg / mL; the volume concentration of DMSO during co-incubation is 5%; the co-incubation temperature is 4℃ and the time is 24 hours. Step S5, Half-life extension modification: IgBD-Cys and DSPE-PEG2000-MAL are mixed to prepare suspension E; the molar ratio of IgBD-Cys to DSPE-PEG2000-MAL is 1:7; a reducing agent is added to suspension E, and suspension F is obtained after incubation; the incubation temperature is 4℃, and the incubation time is 12 hours; the reducing agent is TCEP; the concentration of the reducing agent in suspension E is 5 mM; suspension F is dialyzed to remove free DSPE-PEG2000-MAL to obtain IgBD-DSPE; the semipermeable membrane used for dialyzed suspension F has a molecular weight cutoff of 3. KD; IgBD-DSPE was co-incubated with engineered extracellular vesicles loaded with curcumin to obtain suspension G. Suspension G was dialyzed to remove free protein, yielding IgBD-modified engineered extracellular vesicles, denoted as IgBD-EV-CUR; the co-incubation time was 12 hours; the semipermeable membrane used for dialyzed suspension G had a molecular weight cutoff of 30 KD.

[0030] The reducing agent reduces the disulfide bonds in the protein, allowing the Cys side chain of the protein to fully react with MAL and form stable covalent bonds.

[0031] To use GFP to trace IgBD-EV-CUR, IgBD-GFP-EV-CUR was prepared. The preparation method is as follows: GFP-DSPE was prepared using the same method as IgBD-DSPE. During co-incubation, GFP-DSPE was added to prepare IgBD-GFP-EV-CUR by co-incubating with IgBD-DSPE and EV-CUR. Other parameters were the same as in Example 2.

[0032] Comparative Example 1: Preparation of engineered extracellular vesicles using ultrasound: In step S4 of Example 1, curcumin was loaded into extracellular vesicles using an ultrasonic method to obtain EV-CUR; other steps and parameters were the same as in Example 1. The specific method of ultrasonic loading was as follows: the sample was placed in an ultrasonic machine with an ultrasonic power of 12 W, on / off for 15 s each, reset 3 times, and then placed on ice for 5 minutes. This process was repeated 3 times; after ultrasonication, the mixture was incubated at 37°C for 1 hour.

[0033] Comparative Example 2: Preparation of engineered extracellular vesicles using the repeated freeze-thaw method: In step S4 of Example 1, curcumin was loaded into extracellular vesicles using a repeated freeze-thaw method to obtain EV-CUR; other steps and parameters were the same as in Example 1. The specific method for loading using the repeated freeze-thaw method was as follows: the sample was frozen in a liquid nitrogen tank at -196°C for 1 minute, then thawed at room temperature in the dark for 30 minutes. After thawing, the sample was frozen in a liquid nitrogen tank at -196°C for 1 minute, and this process was repeated 3 times. After the repeated freeze-thaw cycles were completed, the mixture was incubated at 37°C for 1 hour.

[0034] Comparative Example 3, preparation of engineered extracellular vesicles (Ztaq-EV-CUR) by co-incubation method: Ztaq was used instead of IgBD to prepare Ztaq-EV-CUR and Ztaq-GFP-EV-CUR, with other parameters consistent with Example 2.

[0035] The following comparative analysis of in vivo and in vitro systems of the embodiments and comparative examples verifies the effectiveness of the embodiments.

[0036] 1. Effects of different loading methods on curcumin loading Centrifuge the EV-CUR suspension to remove unloaded curcumin; after resuspending, determine the EV-CUR protein concentration using a BCA protein concentration assay kit, expressing the extravesicular protein content as protein mass; the EV-CUR protein concentration was 0.316 μg / μL. Determine the curcumin content and calculate the amount of curcumin loaded per 100 μg EV and the encapsulation efficiency. The amount of curcumin loaded per 100 μg EV is the amount of curcumin loaded per 100 μg vesicle protein; the encapsulation efficiency refers to the mass percentage of curcumin successfully encapsulated within the vesicles relative to the total curcumin dosage.

[0037] The curcumin loading rates for different loading methods are shown in the table below. The co-incubation method yielded the highest loading rate per unit of exovesicle.

[0038]

[0039] 2. Effects of EV-CUR prepared in Example 1 on cellular inflammation in vitro. To investigate the effects of curcumin-loaded extracellular vesicles on inflammation, a cellular inflammation model was constructed using LPS. Extracellular vesicles, curcumin, and curcumin-loaded extracellular vesicles (EV-CUR prepared in Example 1) were then co-incubated with inflammatory cells (A549, BEAS-2B, and MLE-12 cells), respectively. After 24 hours, cells from each group were collected, and the expression levels of inflammatory cytokines IL8, IL6, IL1β, and TNFα were detected. The alleviating effects of extracellular vesicles, curcumin, and curcumin-loaded extracellular vesicles on inflammation in different cells were compared.

[0040] This study used three lung disease-related cell lines: MLE-12, A549, and BEAS-2B, which are widely used in inflammation research. MLE-12 cells, as a mouse lung epithelial cell line, can be used to simulate the inflammatory response of alveolar epithelium; A549 cells are human alveolar basal epithelial adenocarcinoma cells with good adherent growth characteristics, making them a commonly used model for studying lung inflammation and immune responses; BEAS-2B cells are a normal human bronchial epithelial cell line, which can well reflect the airway inflammatory process. By using these three cell lines in combination, the expression of inflammatory factors was comprehensively assessed, providing a reliable in vitro experimental model for in-depth research on the inflammatory mechanisms of acute lung injury.

[0041] 2.1 Methods for constructing cellular inflammation models (1) Take 10 mg of LPS powder (lipopolysaccharide, the main component of the cell wall of Gram-negative bacteria), dissolve the powder in 1 mL of sterile distilled water, and prepare a 10 mg / mL LPS solution.

[0042] (2) Resuscitate MLE-12 cells, BEAS-2B cells and A549 cells respectively, and spread them evenly in a 6-well plate.

[0043] (3) After cell adhesion, cells were randomly divided into two regions. The control group and the LPS group were treated. The LPS solution was diluted to a working solution of 1 mg / mL. 50 μL of 1 mg / mL LPS was added to each well in the LPS group. Treatment lasted 24 hours. Cell RNA was extracted, and the expression levels of inflammatory factors IL8, IL6, IL1β, and TNFα were detected using real-time quantitative PCR (qPCR).

[0044] 2.2 The experimental grouping method is shown in the table below:

[0045] 2.3 Test Results Test results Figure 1 As shown, in A549, BEAS-2B, and MLE-12 cells, the expression levels of inflammatory cytokines IL8, IL6, IL1β, and TNFα significantly increased after LPS treatment, indicating successful model construction. Treatment with EV-CUR prepared in Example 1 significantly reduced the expression levels of inflammatory cytokines IL8, IL6, IL1β, and TNFα, indicating that curcumin-loaded extracellular vesicles can effectively alleviate LPS-induced cellular inflammation.

[0046] 3. Effects of EV-CUR prepared in Example 1 on a mouse model of acute lung injury and inflammation. LPS-induced acute lung injury in mice is an effective model for studying acute lung injury, as it can simulate pathological events such as inflammation and histological changes. Therefore, we used an LPS-induced acute lung injury model in mice for our experiments.

[0047] 3.1 Mouse model of acute lung injury and inflammation Experimental animals were male C57BL / 6 mice from Beijing Vital Rivers, housed in an SPF-grade animal facility. The mice lived in suitable conditions, and their food and drinking water were sterilized using an autoclave. All experiments were conducted in accordance with the animal ethics and guidelines of the Experimental Animal Committee of Xinxiang Medical University. An acute lung injury inflammation model was established in mice by intranasal instillation of 5 mg / kg LPS working solution (concentration 1 mg / mL, not exceeding 50 μL per mouse).

[0048] 3.2 Test Methods Mice were randomly divided into three groups: a negative control group, a positive control group, and a model group. The negative control group consisted of normal mice, which were administered a corresponding dose of physiological saline via nasal drip. The positive control group consisted of mice with an acute lung injury-inflammation model. The model group consisted of mice with an acute lung injury-inflammation model, which were injected with EV-CUR (Example 1) at a dose of 100 μg / mouse via tail vein injection. Four days after administration, the alveoli of the mice were perfused, and lung tissue was analyzed pathologically.

[0049] The experimental grouping method is shown in the table below.

[0050]

[0051] 3.3 Cell count in bronchoalveolar lavage fluid After euthanasia, the mice were dissected using scissors to open the thoracic cavity and expose the larynx. An irrigation needle was inserted into the mouse's larynx, and after the needle was secured, air was injected into the mouse's lungs. The lungs were observed to see if they expanded or leaked air. Subsequently, 1 mL of physiological saline was injected into the mouse's lungs, and the bronchoalveolar lavage fluid was aspirated after gentle compression of the lungs. 10 μL of the lavage fluid was added to a cell counting chamber to count the white blood cells in the bronchoalveolar lavage fluid.

[0052] Centrifuge the remaining rinsing solution at low speed and collect the supernatant. Take 25 μL and measure its absorbance using a protein concentration assay kit. Calculate the corresponding protein concentration.

[0053] 3.4 Pathological analysis of lung tissue (1) Lung tissue collection: 0.2 mL / 20 g of 1% pentobarbital was injected intraperitoneally into the experimental mice to anesthetize them. The mice were then perfused with physiological saline and their lung tissue was collected. The left lung was fixed in 4% paraformaldehyde fixative, and the right lung was divided into three parts and stored in a -80℃ freezer for subsequent experiments.

[0054] (2) Paraffin embedding and sectioning: After the left alveoli was fixed in fixative for 48 hours, it was removed and placed in an embedding cassette, and rinsed with running water for 24 hours. The tissue was then sequentially treated with 70% ethanol overnight, 80% ethanol for 1 hour, 80% ethanol for 1 hour, 90% ethanol for 1 hour, 95% ethanol for 30 minutes, 95% ethanol for 30 minutes, 100% ethanol for 30 minutes, 1 / 2 xylene for 15 minutes, xylene for 10 minutes, xylene for 7 minutes, liquid paraffin for 1 hour, liquid paraffin for 1 hour, and liquid paraffin for 1 hour. Finally, the tissue was placed in an embedding cassette containing molten paraffin, perfused with liquid paraffin, and allowed to solidify at room temperature after embedding. The samples were sectioned using a paraffin microtome, with three 4μm sections cut from each sample along the largest surface of the tissue.

[0055] (3) H&E staining: Place the paraffin sections in a 60℃ constant temperature incubator for 30 min to remove excess paraffin from the sections; then treat them sequentially with xylene for 15 min, xylene for 15 min, 100% ethanol for 5 min, 100% ethanol for 5 min, 95% ethanol for 5 min, 90% ethanol for 5 min, 80% ethanol for 5 min, 70% ethanol for 5 min, distilled water for 5 min, and distilled water for 5 min.

[0056] Add hematoxylin staining solution and react with the slide for 10 seconds, then rinse with a slow stream of water and soak in distilled water for 2 minutes.

[0057] Add eosin staining solution and react with the slide for 30 seconds, then rinse with a slow stream of water and soak in distilled water for 2 minutes.

[0058] Paraffin sections were sequentially dehydrated by 75% ethanol for 3 seconds, 85% ethanol for 3 seconds, 95% ethanol for 3 seconds, and ethanol for 3 seconds, and then soaked twice in xylene for 2 minutes to make the sections transparent.

[0059] Using neutral resin to mount the slides, and after the resin dries, observe and photograph them under a microscope to analyze the pathological changes in the lung tissue.

[0060] (4) Immunohistochemical staining Place the paraffin sections in a 60℃ constant temperature incubator for 30 min to remove excess paraffin. Then, sequentially treat the treated paraffin sections with xylene for 15 min, xylene for 15 min, 100% ethanol for 5 min, 100% ethanol for 5 min, 95% ethanol for 5 min, 90% ethanol for 5 min, 80% ethanol for 5 min, 70% ethanol for 5 min, distilled water for 5 min, and distilled water for 5 min.

[0061] Antigen retrieval is performed on tissue sections using methods such as hot water baths or microwave heating, making the antigens easier to expose during the staining process.

[0062] Tissues are treated with 5-10% bovine serum albumin or other blocking agents to prevent nonspecific antibody binding.

[0063] Primary antibody incubation: The diluted primary antibody (specific antibody) is added to the tissue section to bind to the protein to be detected. Incubation conditions vary depending on experimental requirements, and are usually carried out at room temperature or in a 4°C refrigerator for several hours or overnight.

[0064] Washing: Wash the tissue sections multiple times with washing buffer (e.g., 1×PBS) to remove unbound primary antibodies.

[0065] Secondary antibody incubation: A fluorescently labeled secondary antibody (with a different host animal species than the primary antibody) is added to the tissue section and binds to the primary antibody. The incubation conditions are similar to those for the primary antibody.

[0066] Washing: Tissue sections were washed multiple times with washing buffer to remove unbound secondary antibody.

[0067] Staining: Depending on the experimental requirements, use appropriate staining agents (such as DAB) to stain tissue sections to visualize the expression location of the target protein.

[0068] Reverse staining: If necessary, perform background reverse staining to reduce non-specific staining.

[0069] Dehydration and mounting: The stained tissue sections are dehydrated and then mounted on a glass slide using a transparent medium (such as linoleic acid).

[0070] Observation and analysis: Observe the stained tissue sections under an optical microscope, record or photograph the images, and perform quantitative and qualitative analysis.

[0071] 3.5 Statistical Analysis Statistical analysis was performed using GraphPad Prism version 8. Pairwise comparisons were performed using t-tests. One-way ANOVA was used for inter-group statistical comparisons, employing either the Tukey test or the Dunnett test. A p-value < 0.05 was considered statistically significant; * indicates p < 0.05, ** indicates p < 0.005, *** indicates p < 0.001, and **** indicates p < 0.0001. Data are expressed as arithmetic mean ± standard error of the mean (SEM).

[0072] 3.6 Test Results The test results are shown in Figure 2 .like Figure 2 A, Figure 2 As shown in Figure B, compared to the negative control group, the total protein and white blood cell count in the bronchoalveolar lavage fluid of mice in the positive control group were significantly increased, indicating successful model establishment. Compared to the positive control group, the total protein and white blood cell count in the bronchoalveolar lavage fluid of mice in the model group showed a significant decreasing trend. Figure 2 C Figure 2 D、 Figure 2 E, Figure 2 As shown in Figure F, the model group effectively reduced inflammatory infiltration in the lungs of mice. This indicates that EV-CUR can effectively reduce lung inflammation induced by LPS in mice and alleviate the damage caused by LPS to the lungs of mice.

[0073] 4. The binding of modified proteins IgBD and Ztaq to extracellular vesicles was detected using urea-SDS-PAGE gel electrophoresis and Coomassie brilliant blue staining.

[0074] like Figure 3 As shown, under the action of DSPE-PEG2000-MAL, both IgBD-EV-CUR prepared in Example 2 and Ztaq-EV-CUR prepared in Comparative Example 3 were able to bind to the surface of extracellular vesicles.

[0075] 5. Half-life of IgBD-GFP-EV-CUR 5.1 EV Half-Life Detection Method The IgBD-GFP-EV-CUR prepared in Example 2 and the Ztaq-GFP-EV-CUR prepared in Comparative Example 3 were injected into mice via the tail vein, respectively. At 5, 30, 60, 360, 720, and 1440 minutes after administration, small amounts of blood were collected from the posterior venous vein of the mice using glass tubes. The blood samples were allowed to stand at room temperature for several hours to allow for natural clotting and serum separation. The samples were then centrifuged at 2000 rpm for 10 minutes, and the supernatant was collected as serum. The fluorescence intensity of GFP in the serum was detected, and the measured values ​​were plotted and analyzed. The half-life of extracellular vesicles modified with different proteins in mice was calculated.

[0076] 5.2 Test Results The aggregation of engineered extracellular vesicles in mice was determined by detecting the fluorescence intensity of GFP in serum. Figure 4 As shown, the half-life of IgBD-GFP-DSPE-EV-CUR is 627.9 min, while that of Ztaq-GFP-DSPE-EV-CUR is 10.24 min. This result indicates that IgBD modification can significantly increase the residence time of extracellular vesicles in mice and prolong their half-life.

[0077] 6. Effects of IgBD modification on exovesicle phenotype To detect the phenotypic changes of IgBD-modified exovesicles, the particle size and phenotypic of engineered exovesicles (EV obtained in step S3 of Example 1, EV-CUR obtained in step S4 of Example 1, and IgBD-EV-CUR prepared in Example 2) were measured.

[0078] Extracellular vesicles obtained by low-temperature ultracentrifugation were diluted with 1×PBS to prepare a suspension, and the particle size distribution of the extracellular vesicles in the suspension was detected using nanoflow cytometry. Figure 5 As shown, the particle size of the extracellular vesicles is approximately 54 nm, which is consistent with the particle size characteristics of extracellular vesicles.

[0079] Transmission electron microscopy was used to observe whether the morphology of the modified extracellular vesicles changed. For example... Figure 6 As shown, the extracellular vesicles are intact and the double membrane structure is clearly visible, exhibiting a disc-shaped structure.

[0080] The above results indicate that IgBD-modified extracellular vesicles showed no significant changes in particle size or phenotype, and that IgBD modification does not affect the phenotype of extracellular vesicles.

[0081] 7. Effects of IgBD modification on the aggregation of extracellular vesicles in the lungs 7.1 Test Methods To detect the aggregation of IgBD-modified extracellular vesicles in the lungs, engineered extracellular vesicles (EV-CUR prepared in Example 1 and IgBD-EV-CUR prepared in Example 2) were injected into mice via the tail vein. Three hours after administration, lung tissue was harvested from the mice. The fluorescence intensity of the aggregation in the lungs of each group of mice was detected using in vivo imaging.

[0082] 7.2 The in vivo imaging method is as follows: (1) Add 3 μL of DiR (25 mM) to 1500 μL of EV (1 μg / μL) and mix well. Wrap the EP tube with aluminum foil to achieve a light-protected environment. Then place it in a cell incubator and incubate at 37°C for 30 min in the dark.

[0083] (2) Transfer the incubated solution to an ultracentrifuge tube and centrifuge at 120,000 × g for 2 h to remove free excess dye. Resuspend in 1×PBS buffer to obtain DiR-labeled EV.

[0084] (3) When using DiR-labeled EV instead of EV to prepare engineered exovesicles (EV-CUR prepared in Example 1 and IgBD-EV-CUR prepared in Example 2), DiR-labeled EV-CUR and IgBD-EV-CUR are obtained.

[0085] (4) The mice were randomly divided into three groups of three. The engineered extravesicles and control saline were injected into the corresponding mice via the tail vein.

[0086] (5) Three hours after administration, lung tissue was removed from each group of mice. The fluorescence intensity of the accumulated cells in the lungs of each group of mice was detected by in vivo imaging.

[0087] 7.3 Test Results like Figure 7 , Figure 8 As shown, the fluorescence accumulation in the lungs of mice in the EV-CUR group was higher than that in the control group, indicating that loading curcumin onto extracellular vesicles can enhance curcumin accumulation in the lungs. The fluorescence accumulation in the lungs of mice in the IgBD-EV-CUR group was higher than that in the EV-CUR group, indicating that IgBD modification can further enhance the accumulation of extracellular vesicles in the lungs.

[0088] 8. Effects of IgBD-modified extracellular vesicles on inflammation in MLE-12 cells The inhibitory effect of engineered extracellular vesicles on inflammation was examined at the cellular level. An MLE-12 cell inflammation model was constructed using LPS. EV-CUR and IgBD-EV-CUR engineered extracellular vesicles were co-incubated with inflammatory cells to assess the alleviating effect of extracellular vesicles on cellular inflammation.

[0089] like Figure 9 As shown, engineered extracellular vesicles can inhibit LPS-induced inflammation in MLE-12 cells, and the long half-life modification with IgBD can further enhance the inhibitory effect of extracellular vesicles on cell inflammation.

[0090] 9. Effects of different modified extracellular vesicles on acute lung injury in mice To examine the inhibitory effect of engineered extracellular vesicles on inflammation at the animal level, an acute lung injury model was constructed in mice using LPS. Physiological saline (represented as control), LPS working solution (represented as LPS), EV-CUR, and IgBD-EV-CUR were injected into mice via the tail vein. Four days later, bronchoalveolar lavage fluid was collected, and the total protein and white blood cell counts in the bronchoalveolar lavage fluid of different groups of mice were measured. Lung tissue was also collected to observe lung morphology, and the expression of inflammatory factors in the lungs of different groups of mice was measured.

[0091] like Figure 10 , Figure 11 As shown, LPS treatment significantly increased the total protein content and white blood cell count in the bronchoalveolar lavage fluid of mice, and significantly increased the levels of inflammatory factors IL8, IL6, IL1β, and TNFα. After EV-CUR treatment, the total protein and white blood cell count in the bronchoalveolar lavage fluid of mice decreased, and the levels of pulmonary inflammatory factors IL8, IL6, IL1β, and TNFα were significantly reduced. After IgBD-EV-CUR treatment, the total protein and white blood cell count in the bronchoalveolar lavage fluid of mice decreased further, and the expression levels of pulmonary inflammatory factors IL8, IL6, IL1β, and TNFα were further reduced.

[0092] like Figure 12-15 As shown, LPS induction induces inflammatory infiltration in the lungs of mice and increases CD68 secretion. EV-CUR treatment effectively reduces inflammatory infiltration in the lungs and decreases CD68 secretion. IgBD-EV-CUR can further reduce inflammatory infiltration in the lungs and decrease CD68 secretion.

[0093] In summary, EV-CUR and IgBD-EV-CUR can inhibit lung inflammation and improve and treat acute lung injury.

Claims

1. An engineered extracellular vesicle, characterized in that, The engineered extracellular vesicles are modified with IgBD and loaded with active ingredients.

2. The engineered extracellular vesicles according to claim 1, characterized in that, The active ingredient is curcumin.

3. The engineered extracellular vesicles according to claim 2, characterized in that, The engineered extracellular vesicles have a curcumin loading greater than 1.6 μg / 100 μg.

4. A method for preparing engineered extracellular vesicles, characterized in that, Includes the following steps: Step S1, collect cell culture supernatant: after mesenchymal stem cells are cultured in complete culture medium, washed, and cultured in basal culture medium, collect the basal culture medium supernatant A; Step S2, Removal of cell debris: Cell debris is removed from supernatant A by centrifugation to obtain supernatant B; Step S3, Preparation of extracellular vesicles: Centrifuge the supernatant B and collect the precipitate C; Precipitate C was washed and resuspended to obtain extracellular vesicle suspension D; Step S4, Loading the active ingredient: Mix the extracellular vesicle suspension D with the active ingredient solution evenly, and load the active ingredient into the extracellular vesicles using a co-incubation method; the active ingredient is curcumin; Step S5, Protein Modification: Engineered extracellular vesicles loaded with active ingredients are modified with IgBD to form engineered extracellular vesicles.

5. A pharmaceutical preparation, characterized in that, The pharmaceutical formulation comprises the engineered extracellular vesicles as described in claim 1.

6. The use of the engineered extracellular vesicles as described in claim 1 in a pharmaceutical formulation for the treatment of acute lung injury.

7. The application of the engineered extracellular vesicles as described in claim 1 in a drug delivery system.

8. A method for increasing the loading of curcumin in extracellular vesicles, characterized in that, The method described herein employs the preparation method as described in claim 4.

9. A method for prolonging the half-life of extracellular vesicles, characterized in that, The method described herein employs the preparation method as described in claim 4.

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