An engineered extracellular vesicle

Abstract

The application relates to the technical field of biological medicine, and particularly discloses an engineered extracellular vesicle, which is derived from M2 type macrophages, and is coupled with M1pep homing targeting peptides on the surface and loaded with S1PR2 inhibitor JTE-013 in the inside. The M2 type macrophage-derived extracellular vesicle has natural inflammatory chemotaxis and immune escape characteristics; the M1pep homing targeting peptide can accurately recognize and combine with M1 type macrophages, and the M1pep homing targeting peptide and the M2 type macrophage-derived extracellular vesicle jointly form a double-targeting mechanism of inflammatory tissue chemotaxis and cell recognition; after the JTE-013 loaded in the inside is released in a targeted mode, the JTE-013 can block the S1P-S1PR2 signal channel, and cooperates with the endogenous anti-inflammatory signal in the vesicle to induce the conversion of macrophages from M1 type to M2 type, so that the inflammatory reaction is effectively inhibited, and the edema and necrosis caused by acute pancreatitis and secondary lung tissue damage are improved.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an engineered extracellular vesicle. Background Technology

[0002] Acute pancreatitis (AP) is a sudden inflammatory disease of the pancreas, primarily caused by the abnormal activation of pancreatic enzymes within the pancreas, leading to acute inflammation of the pancreas itself and surrounding tissues. Most patients present with mild, self-limiting acute pancreatitis, usually resolving within one week. However, approximately 20% can progress to moderate to severe acute pancreatitis, accompanied by pancreatic necrosis or organ failure, with a mortality rate as high as 20%–40%. Current treatment is primarily supportive, including fluid resuscitation, pain management, and nutritional support; specific therapies targeting the core pathophysiological mechanisms of the disease are still lacking.

[0003] In the progression of acute pancreatitis, sphingosine-1-phosphate (S1P), a bioactive lipid mediator produced by sphingosine kinase, plays a crucial role in inflammation regulation by activating various downstream signaling pathways through binding to its receptor S1PR2. When pancreatic tissue is damaged, the metabolism of S1P released by acinar cells is abnormal, and its expression level is elevated. After binding to S1PR2 on the surface of macrophages, this molecule can promote macrophage polarization towards the pro-inflammatory M1 phenotype, thereby promoting the secretion of large amounts of inflammatory factors and forming a positive feedback loop that exacerbates acinar cell damage. Therefore, targeting the S1P / S1PR2 signaling axis between damaged acinar cells and M1 macrophages has become a potential therapeutic strategy for acute pancreatitis. JTE-013, as a selective S1PR2 inhibitor, can effectively block the downstream S1P / S1PR2 signaling pathway and theoretically inhibit the inflammatory cycle in acute pancreatitis. However, this drug suffers from poor cell targeting and insufficient immune regulation, which greatly limits its therapeutic efficacy and practical application. Summary of the Invention

[0004] To address the issues of poor targeting and insufficient immune regulation of S1PR2 inhibitors in the treatment of acute pancreatitis, this invention proposes an engineered extracellular vesicle. This engineered extracellular vesicle is based on extracellular vesicles derived from M2 macrophages, and after engineering modification, its surface is coupled with the M1pep homing target peptide, and its interior is loaded with an S1PR2 inhibitor. Among them, extracellular vesicles derived from M2 macrophages, in addition to their natural inflammatory chemotaxis, also possess immune escape properties as homologous cell membranes; the M1pep homing target peptide can accurately recognize and bind to M1 macrophages, forming a dual targeting mechanism of inflammatory tissue chemotaxis and M1 macrophage recognition together with extracellular vesicles derived from M2 macrophages; after targeted release of JTE-013 loaded internally via exocytosis, it can effectively block the S1P-S1PR2 signaling pathway and synergize with the inherent anti-inflammatory function of extracellular vesicles derived from M2 macrophages, promoting the transformation of macrophages from M1 to M2, thereby reducing inflammatory storms, alleviating the progression of acute pancreatitis, and achieving safe targeted therapy for acute pancreatitis.

[0005] Based on the above findings, the present invention provides the following technical solution: In a first aspect, the present invention provides an engineered extracellular vesicle derived from an M2 macrophage, having an M1pep homing-targeting peptide coupled to its surface and an S1PR2 inhibitor loaded internally; the amino acid sequence of the M1pep homing-targeting peptide is shown in SEQ ID NO:1.

[0006] In conjunction with the first aspect, in some embodiments, the M1pep homing-targeting peptide is coupled to the surface of the extracellular vesicles via a DSPE-PEG-Mal linker.

[0007] In conjunction with the first aspect, in some embodiments, the S1PR2 inhibitor is one or more of JTE-013, GLPG2938, and AB1.

[0008] Secondly, the present invention provides a method for preparing engineered extracellular vesicles, comprising the following steps: Provides extracellular vesicles derived from M2 macrophages; The extracellular vesicles were co-incubated with the S1PR2 inhibitor to obtain extracellular vesicles loaded with the S1PR2 inhibitor. The M1pep homing-targeting peptide was reacted with DSPE-PEG-Mal linker to obtain DSPE-PEG-M1pep; The engineered extracellular vesicles were obtained by co-incubating the DSPE-PEG-M1pep with the extracellular vesicles loaded with the S1PR2 inhibitor.

[0009] In conjunction with the second aspect, in some embodiments, providing M2 macrophage-derived extracellular vesicles includes: Macrophage M2 polarization was induced by anti-inflammatory cytokines, and cell culture supernatant was collected. Extracellular vesicles derived from M2 macrophages were collected from the cell culture supernatant by differential centrifugation.

[0010] In conjunction with the second aspect, in some embodiments, the S1PR2 inhibitor is JTE-013, and the concentration of JTE-013 is 50~150 μg / mL.

[0011] In conjunction with the second aspect, in some embodiments, the concentration of extracellular vesicles derived from M2 macrophages is 0.5 to 2 mg / mL.

[0012] In conjunction with the second aspect, in some embodiments, the mass ratio of DSPE-PEG-M1pep to extracellular vesicles loaded with JTE-013 is 1:(10~50).

[0013] Thirdly, the present invention provides the application of the above-mentioned engineered extracellular vesicles in the preparation of a medicament for treating acute pancreatitis.

[0014] In conjunction with the third aspect, in some embodiments, the drug comprises engineered extracellular vesicles and their pharmaceutically acceptable carriers.

[0015] Compared with the prior art, the present invention has at least the following beneficial effects: (1) The engineered extracellular vesicles provided by this invention fully utilize the inherent inflammatory chemotactic properties of M2 macrophage vesicles and combine them with the active targeting ability of M1pep peptides. They can efficiently accumulate in the site of pancreatic inflammation and around inflammatory macrophages, thereby achieving precise intervention on the inflammatory microenvironment of acute pancreatitis. As a natural drug carrier, these vesicles have good biocompatibility and low immunogenicity, which can significantly improve the stability and safety of drugs in vivo and reduce systemic side effects. In vivo experiments show that, compared with the use of JTE-013 alone, these engineered extracellular vesicles can significantly improve pathological damage such as pancreatic edema and necrosis in acute pancreatitis model mice, alleviate lung tissue damage, and reduce serum amylase, lipase, and inflammatory factors such as TNF-α, IL-1β, and IL-6, effectively inhibiting the inflammatory response.

[0016] (2) This engineered extracellular vesicle can release JTE-013 via exocytosis, thereby blocking the S1P-S1PR2 signaling pathway and inhibiting macrophage polarization towards the pro-inflammatory M1 phenotype. Simultaneously, the anti-inflammatory factors carried by the M2-derived extracellular vesicles can also synergistically reprogram macrophages, alleviating pancreatic tissue damage. In vitro mechanism experiments show that this engineered extracellular vesicle can also effectively inhibit macrophage M1 polarization and the release of inflammatory factors by inhibiting signaling pathways such as PI3K, NLRP3, and AMPK. Compared with traditional single-pathway supportive therapy, the engineered extracellular vesicles proposed in this invention can exert anti-inflammatory effects through multiple mechanisms, effectively reshaping the inflammatory microenvironment of acute pancreatitis. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 The physicochemical properties and stability assessment of JTE-013@M1pep-M2MPs were performed, including: (A) the total protein content on the surface of the M2MPs membrane was detected by BCA method; (B) the expression of CD-206-APC on the surface of the M2MPs membrane was detected by flow cytometry, where ** indicates p<0.01, *** indicates p<0.001, and **** indicates p<0.0001; (C) the effective modification ratio of M1pep in M1pep-M2MPs was determined by flow cytometry, where ** indicates p<0.01, *** indicates p<0.001, and **** indicates p<0.0001; and (D) the JTE-013 content in JTE-013@M1pep-M2MPs was evaluated by high performance liquid chromatography. The loading capacity; (E~F) The particle size changes of M2MPs, M1pep-M2MPs, JTE-013@M2MPs and JTE-013@M1pep-M2MPs at different time points were detected by NTA analysis in PBS solution or PBS solution containing 10% FBS; (G~H) The changes in surface potential of M2MPs, M1pep-M2MPs, JTE-013@M2MPs and JTE-013@M1pep-M2MPs at different time points were evaluated by zeta potential analyzer in PBS solution or PBS solution containing 10% FBS; (I) The effect of different concentrations of free JTE-013 on the activity and marker expression of M1 macrophages.

[0019] Figure 2The study included: in vitro targeting and in vivo distribution verification of JTE-013@M1pep-M2MPs; (A) Zeta potential analysis was used to detect the zeta potentials of M2MPs, M1pep-M2MPs, JTE-013@M2MPs, and JTE-013@M1pep-M2MPs; (B) NTA analysis was used to detect the particle size distribution of M2MPs, M1pep-M2MPs, JTE-013@M2MPs, and JTE-013@M1pep-M2MPs; (C) Confocal fluorescence microscopy was used to analyze the M2MP loading efficiency of M1pep coupled with Cy5 fluorescence; (D) Electron microscopy was used to examine the M2MPs, M1pep-M2MPs, and JTE-013@M1pep-M2MPs. Morphological schematic diagrams of @M2MPs and JTE-013@M1pep-M2MPs; (E~G) Image results of M1pep-M2MPs versus M2MPs targeting M0, M1, and M2 macrophages using confocal microscopy, and quantitative analysis of corresponding fluorescence intensity and the number of DIO fluorescently labeled particles; (H~I) Image and quantitative analysis of the targeting efficiency of M1pep-M2MPs versus M2MPs on lesion tissue in an acute pancreatitis mouse model using in vivo fluorescence imaging, where ** indicates p<0.01, *** indicates p<0.001, and **** indicates p<0.0001.

[0020] Figure 3 The study aimed to assess the in vitro safety of JTE-013@M1pep-M2MPs, including: (A) Calcein-AM / PI double staining method to evaluate the effects of JTE-013@M1pep-M2MPs at concentrations of 0 μg / mL, 5 μg / mL, 15 μg / mL, and 50 μg / mL on the activity of immortalized pancreatic stellate cells, pancreatic acinar carcinoma cells, and bone marrow-derived macrophages after treatment for 24, 48, and 72 hours, respectively; (B) Quantitative analysis of the cell activity of pancreatic stellate cells after treatment with different concentrations of JTE-013@M1pep-M2MPs for different durations; (C) Quantitative analysis of the cell activity of pancreatic acinar carcinoma cells after treatment with different concentrations of JTE-013@M1pep-M2MPs for different durations; and (D) Quantitative analysis of the cell activity of bone marrow-derived macrophages after treatment with different concentrations of JTE-013@M1pep-M2MPs for different durations.

[0021] Figure 4The in vivo safety assessment of JTE-013@M1pep-M2MPs included: (A) evaluating the effect of HE staining on the pathological damage of JTE-013@M1pep-M2MPs on the heart, liver, spleen, lung, kidney, and pancreas tissues of normal mice; (B) detecting the changes in the level of amylase in the serum of normal mice after treatment with JTE-013@M1pep-M2MPs; (C) detecting the changes in the level of lipase in the serum of normal mice after treatment with JTE-013@M1pep-M2MPs; and (D) detecting the level of alanine aminotransferase (ALT) in the serum of normal mice after treatment with JTE-013@M1pep-M2MPs. (E) Changes in the level of aspartate aminotransferase (AST) in the serum of normal mice after treatment with JTE-013@M1pep-M2MPs; (F) Changes in the level of albumin (ALB) in the serum of normal mice after treatment with JTE-013@M1pep-M2MPs; (G) Changes in the level of alkaline phosphatase (ALP) in the serum of normal mice after treatment with JTE-013@M1pep-M2MPs; (H) Changes in the level of blood urea nitrogen (BUN) in the serum of normal mice after treatment with JTE-013@M1pep-M2MPs; (I) Changes in the level of creatinine (CRE) in the serum of normal mice after treatment with JTE-013@M1pep-M2MPs.

[0022] Figure 5 Evaluation of the effect of JTE-013@M1pep-M2MPs on RAW264.7 cell polarization; including: (A) Flowchart of the co-culture experiment of pancreatic acinar cells and macrophages; (B) CM CCK Under stimulation, different treatments such as JTE-013@M1pep-M2MPs affected MHC II in macrophages. + The effect of cell ratio; (C)CM CCK Under stimulation, different treatments such as JTE-013@M1pep-M2MPs affected CD80 in macrophages. + Effects on cell proportions; (D) Effects of different treatments such as JTE-013@M1pep-M2MPs on MHC II in macrophages under S1P stimulation. + Effects on cell proportions; (E)S1P stimulation, different treatments such as JTE-013@M1pep-M2MPs on CD80 in macrophages +Effects on cell proportions; (F) Effects of different treatments such as JTE-013@M1pep-M2MPs on CD86 expression levels in macrophages under S1P stimulation; (G) Effects of different treatments such as JTE-013@M1pep-M2MPs on TNF-α expression levels in macrophages under S1P stimulation; (H) Effects of different treatments such as JTE-013@M1pep-M2MPs on MRC1 expression levels in macrophages under S1P stimulation; (I) Effects of different treatments such as JTE-013@M1pep-M2MPs on Arg1 expression levels in macrophages under S1P stimulation; (J) Immunoblotting detection of phosphorylation (p-) of PI3K, P38, JNK, and ERK in macrophages of each group. Expression of PI3K, p-P38, p-JNK, p-ERK), total protein (t-PI3K, t-P38, t-JNK, t-ERK), and NLRP3; (K) Quantitative analysis of the relative expression level of p-PI3K protein in macrophages of each group; (L) Quantitative analysis of the relative expression level of p-P38 protein in macrophages of each group; (M) Quantitative analysis of the relative expression level of p-JNK protein in macrophages of each group; (N) Quantitative analysis of the relative expression level of p-ERK protein in macrophages of each group; (O) Quantitative analysis of the relative expression level of NLRP3 protein in macrophages of each group. ** indicates p<0.01, *** indicates p<0.001, **** indicates p<0.0001.

[0023] Figure 6 JTE-013@M1pep-M2MPs significantly improved lecithin-induced acute pancreatitis in mice; among them: (A) Flowchart of the experimental procedure for constructing an acute pancreatitis model mouse using lecithin; (B) HE staining results of pancreatic tissues of mice in each group (200 μm); (C) HE staining results of lung tissues of mice in each group (50 μm); (D) Immunohistochemical staining results of CD45 immunocellular infiltration in pancreatic tissues of mice in each group (100 μm); (E) Immunohistochemical staining results of MPO immunocellular infiltration in pancreatic tissues of mice in each group (100 μm). (μm); (F) Quantitative statistics of pathological damage scores of pancreatic and lung tissues in each group of mice; (G) Quantitative statistics of pathological damage scores of lung tissues in each group of mice; (H) Quantitative statistics of pancreatic weight / body weight ratio in each group of mice; (I) Expression level of amylase in serum of each group of mice; (J) Expression level of lipase in serum of each group of mice; (K) Immunohistochemical detection of CD45 in pancreatic tissues of each group of mice. + Percentage of positive white blood cells; (L) Immunohistochemical detection of MPO in pancreatic tissue of mice in each group +Percentage of neutrophil-positive cells; (M) Serum TNF-α expression level in each group of mice; (N) Serum IL-1β expression level in each group of mice; (O) Serum IL-6 expression level in each group of mice. ** indicates p<0.01, *** indicates p<0.001, **** indicates p<0.0001.

[0024] Figure 7 JTE-013@M1pep-M2MPs reprogrammed macrophages in acute pancreatitis tissue; including: (A) Immunofluorescence colocalization analysis of macrophages (F4 / 80 labeled purple fluorescence) and M1 macrophages (CD86 labeled green fluorescence) in pancreatic tissue; (B) Immunofluorescence colocalization analysis of macrophages (F4 / 80 labeled purple fluorescence) and M2 macrophages (CD206 labeled green fluorescence) in pancreatic tissue; (C~D) Immunohistochemical staining results of p-NF-κB and SPHK1 in mouse pancreatic tissue; (E) F4 / 80 in the immunofluorescence experiment. + CD86 + Quantitative analysis of the proportion of positive cells; (F) F4 / 80 in the above immunofluorescence experiment. + CD206 + Quantitative analysis of the proportion of positive cells; (G~H) Quantitative analysis of the proportion of p-NF-κB and SPHK1 positive regions in the above immunohistochemical experiments; (I) S1P expression level in the serum of mice in each group; (J) Immunoblotting analysis of the expression of IL-1β, IL-6, TNF-α, t-NF-κB, p-NF-κB and SPHK1 in mouse pancreatic tissue; (K) Quantitative analysis of the expression of IL-1β, IL-6, TNF-α, p-NF-κB / t-NF-κB and SPHK1 in the above immunoblotting results, ** indicates p<0.01, *** indicates p<0.001, **** indicates p<0.0001.

[0025] Figure 8The transcriptome was used to evaluate the effect of JTE-013@M1pep-M2MPs on alleviating tissue damage in acute pancreatitis, including: (A) the transcriptome sequencing experimental workflow of pancreatic tissue from mice with acute pancreatitis; (B) a heatmap of differentially expressed genes in pancreatic tissue from the acute pancreatitis model group (AP) and the JTE-013@M1pep-M2MPs treatment group (AP_MPs); (C) principal component analysis (PCA) plot based on the differentially expressed gene heatmap; (D) volcano plot of differentially expressed genes in pancreatic tissue from the AP_MPs group and the AP group; (E) a heatmap of all differentially expressed genes in pancreatic tissue from the AP_MPs group and the AP group; (F) a heatmap of key inflammation-related differentially expressed genes in pancreatic tissue from the AP_MPs group and the AP group. (G) KEGG pathway enrichment analysis of differentially expressed genes in pancreatic tissues of AP_MPs group and AP group; (H) GO functional enrichment analysis of differentially expressed genes in AP_MPs group and AP group; (I) From left to right, the gene expression profile heatmap and trend curve of TNF signaling pathway, NF-κB signaling pathway, pancreatic secretion and protein digestion and absorption pathway; (J~N) qRT-PCR verification of the transcriptional expression levels of Sphk1 (J), Cxcl2 (K), MCP1 (L), CD86 (M) and iNOS (N) in pancreatic tissues of mice in different treatment groups, ** indicates p<0.01, *** indicates p<0.001, **** indicates p<0.0001. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0027] Pancreatic acinar cell damage is the initiating step in the pathogenesis of acute pancreatitis. Under the stimulation of external pathological factors, pancreatic enzymes are abnormally activated and released within pancreatic acinar cells. At the same time, the NF-κB inflammatory signaling pathway is activated, prompting cells to release a large number of inflammatory factors. This process not only leads to pancreatic acinar cell damage but also recruits various immune cells and causes them to interact, triggering a cascade immune response that forms a "cytokine storm," ultimately amplifying the inflammatory response and exacerbating pancreatic acinar cell damage.

[0028] Macrophages are key immune cells in the ongoing damage process of acute pancreatitis, and their number, activation, and polarization patterns determine the severity of acute pancreatitis. Damaged pancreatic acinar cells recruit and polarize macrophages, leading to the accumulation of inflammatory M1 macrophages in the pancreas. These macrophages release large amounts of inflammatory factors such as TNF-α, IL-6, and IL-1β, exacerbating pancreatic acinar cell damage and necrosis, and amplifying the inflammatory response. Therefore, targeting M1 macrophage reprogramming has become a potential therapeutic target for acute pancreatitis.

[0029] M2 macrophage-derived extracellular vesicles (EVs) are promising natural nanotherapeutic agents. Their significant advantage lies in their ability to act as a multi-target immunomodulatory system, effectively addressing excessive inflammatory responses such as cytokine storms. Compared to traditional single-target drugs or glucocorticoids, M2 macrophage-derived EVs carry multiple active ingredients derived from M2 macrophages, enabling them to synergistically inhibit multiple key pro-inflammatory signaling pathways, thus providing more comprehensive inflammation control. Furthermore, M2-EVs exhibit good biocompatibility, low immunogenicity, and excellent tissue targeting capabilities, allowing for efficient uptake by immune cells such as macrophages at the site of inflammation, enhancing therapeutic specificity and reducing systemic side effects.

[0030] This invention provides an engineered extracellular vesicle derived from M2 macrophages, with its surface coupled to an M1pep homing-targeting peptide and its interior loaded with an S1PR2 inhibitor; the amino acid sequence of the M1pep homing-targeting peptide is shown in SEQ ID NO:1. Among them, extracellular vesicles derived from M2 macrophages, in addition to their natural inflammatory chemotaxis, also possess immune escape properties as homologous cell membranes; the M1pep homing target peptide can accurately recognize and bind to M1 macrophages, forming a dual targeting mechanism of inflammatory tissue chemotaxis and M1 macrophage recognition together with extracellular vesicles derived from M2 macrophages; after targeted release of JTE-013 loaded internally via exocytosis, it can effectively block the S1P-S1PR2 signaling pathway and synergize with the inherent anti-inflammatory function of extracellular vesicles derived from M2 macrophages, promoting the transformation of macrophages from M1 to M2, thereby reducing inflammatory storms, alleviating the progression of acute pancreatitis, and achieving safe targeted therapy for acute pancreatitis.

[0031] In some embodiments, the M1pep homing target peptide is coupled to the surface of the extracellular vesicle via a DSPE-PEG-Mal linker. The molecular structure of DSPE-PEG-Mal includes a lipid anchoring group (DSPE), a polyethylene glycol (PEG) chain, and a maleimide reactive group (Mal). DSPE can efficiently insert into the lipid bilayer of the extracellular vesicle, the terminal Mal can specifically covalently couple with the thiol group in the M1pep homing target peptide, and the middle PEG chain can form a hydrophilic protective layer, reducing non-specific protein adsorption, thereby prolonging the in vivo circulation time of the extracellular vesicle, improving its stability, preventing particle aggregation, and thus enhancing its biocompatibility.

[0032] In some embodiments, the S1PR2 inhibitor is one or more of JTE-013, GLPG2938, and AB1. Preferably, the S1PR2 inhibitor is JTE-013. JTE-013 was chosen as the extracellular vesicle loading target because its poor in vivo stability and unfavorable pharmacokinetics severely limit its application, and there is an urgent need to improve and solve these problems with the help of a delivery carrier.

[0033] This invention also provides a method for preparing engineered extracellular vesicles, comprising the following steps: Extracellular vesicles derived from M2 macrophages were extracted; Extracellular vesicles loaded with S1PR2 inhibitor were co-incubated with the inhibitor. The M1pep homing-targeting peptide was reacted with DSPE-PEG-Mal linker to obtain DSPE-PEG-M1pep; Engineered extracellular vesicles were obtained by co-incubating DSPE-PEG-M1pep with extracellular vesicles loaded with S1PR2 inhibitor.

[0034] In some embodiments, providing M2 macrophage-derived extracellular vesicles includes: Macrophage M2 polarization was induced by anti-inflammatory cytokines, and cell culture supernatant was collected. Extracellular vesicles derived from M2 macrophages were collected from cell culture supernatant by differential centrifugation.

[0035] Preferably, the anti-inflammatory cytokine is IL4, with a working concentration of 40 ng / mL.

[0036] In some embodiments, the concentration of JTE-013 is 50-150 μg / mL. Preferably, the concentration of JTE-013 used when co-incubating with extracellular vesicles is 100 μg / mL.

[0037] In some embodiments, the concentration of M2 macrophage-derived extracellular vesicles is 0.5–2 mg / mL. Preferably, the concentration of M2 macrophage-derived extracellular vesicles when co-incubated with JTE-013 is 1 mg / mL.

[0038] In some embodiments, the mass ratio of DSPE-PEG-M1pep to JTE-013-loaded extracellular vesicles during co-incubation is 1:(10~50). Preferably, the mass ratio of DSPE-PEG-M1pep to JTE-013-loaded extracellular vesicles during co-incubation is 1:25.

[0039] This invention also provides the application of the above-mentioned engineered extracellular vesicles in the preparation of drugs for treating acute pancreatitis. In vivo experiments show that these engineered extracellular vesicles can significantly improve pathological damage such as pancreatic edema and necrosis in acute pancreatitis model mice, alleviate lung tissue damage, and reduce serum amylase, lipase, and inflammatory factors such as TNF-α, IL-1β, and IL-6, effectively inhibiting the inflammatory response. In vitro experiments show that these engineered extracellular vesicles, in the conditioned supernatant of pancreatic acinar cancer cells containing natural S1P or in a culture system with exogenously added S1P, can significantly inhibit the expression of CD86 and TNF-α, while promoting the expression of MRC1 and Arg1. Furthermore, they can effectively reverse the upregulation of p-PI3K, p-P38, p-JNK, p-ERK, and NLRP3 protein expression levels induced by S1P stimulation.

[0040] In some embodiments, the drug comprises engineered extracellular vesicles and their pharmaceutically acceptable carriers.

[0041] Unless otherwise specified in the following examples, MPs means uninduced macrophage-derived extracellular vesicles; M2MPs means extracellular vesicles derived from M2 macrophages; M1pep-M2MPs means extracellular vesicles derived from M2 macrophages with M1pep attached to their surface; JTE-013@M2MPs means extracellular vesicles derived from M2 macrophages loaded with JTE-013; JTE-013@M1pep-M2MPs means extracellular vesicles derived from M2 macrophages with M1pep attached to their surface and loaded with JTE-013 internally, which are engineered extracellular vesicles.

[0042] Example 1: Preparation of JTE-013@M1pep-M2MPs (1) Using IL-4 to induce mouse mononuclear macrophage RAW264.7 to polarize into M2 type macrophages. First, IL-4 (purchased from PeproTech) was dissolved in deionized water and prepared into a working solution with a concentration of 40 ng / mL using medium containing 2% FBS. Then, this working solution was added to the culture system of mouse mononuclear macrophage line RAW264.7 to induce M2 macrophage polarization. After 24 hours of treatment, the cells were directly irradiated with ultraviolet light for 1 hour, and then cultured overnight in an incubator.

[0043] (2) Preparation of M2MPs Take the cell culture supernatant of M2 macrophages from (1), centrifuge at 4℃ and 3000 rpm for 10 minutes to remove cell debris. Transfer the supernatant after centrifugation to a new centrifuge tube, centrifuge at 4℃ and 14000 rpm for 30 minutes, discard the supernatant, collect the precipitate and resuspend it in PBS, centrifuge at 4℃ and 14000 rpm for 30 minutes, and finally resuspend it in an appropriate amount of PBS to obtain extracellular vesicles derived from M2 macrophages, which are named M2MPs.

[0044] (3) Preparation of JTE-013@M2MPs Add 100 μg / mL JTE-013 to 1 mg / mL M2MPs, incubate in an incubator for 1 hour, then transfer to a 4°C refrigerator for overnight incubation. The next day, centrifuge at 14000 rpm for 30 minutes and collect the sediment to obtain M2MPs loaded with JTE-013, which is named JTE-013@M2MPs.

[0045] (4) Preparation of DSPE-PEG-M1pep 10 mg of M1pep (purchased from Hefei Guotai Biotechnology) was weighed and dissolved in 0.5 mL of PBS solution with a pH of 7.4. Tris(2-carboxyethyl)phosphine was added to the PBS solution beforehand to a final concentration of 5 mM. Then, according to a molar ratio of distearate-phosphatidylethanolamine-polyethylene glycol-maleimide (DSPE-PEG-Mal) to M1pep of 1:1.2, DSPE-PEG-Mal (purchased from MCE) was weighed and dissolved in 0.5 mL of dimethylformamide. The dissolved M1pep and DSPE-PEG-Mal were added to a round-bottom flask, and PBS with a pH of 8.0 was added to bring the total volume to 12 mL. The mixture was stirred at room temperature for 4 hours. The resulting mixture was then transferred to a dialysis bag with a molecular weight of 3000 and dialyzed in PBS for 0.5 hours, followed by overnight dialyzing with fresh PBS. The next day, the mixture was dialyzed in pure water, with the external pure water changed every 6 hours, until a total of 48 hours of dialyzing was completed. The dialyzed system was transferred to a sterile 50 mL EP tube and frozen into a solid at -20°C. The sample was then transferred to a lyophilizer, weighed, and sterilized by UV irradiation for 1 hour in a clean bench. PBS was added to dissolve the sample to a concentration of 2 mg / mL, yielding the M1pep homing targeting peptide, which was named DSPE-PEG-M1pep. The peptide was then aliquoted and stored at -20°C.

[0046] (5) Preparation of M1pep-M2MPs The concentration of M2MPs was adjusted to 1 mg / mL, and incubated with DSPE-PEG-M1pep at a mass ratio of 25:1 at 37°C for 4 hours. After incubation at 4°C overnight, the precipitate was collected after centrifugation at 14000 rpm for 30 minutes the next day. M2MPs with M1pep coupled to the surface were obtained and named M1pep-M2MPs.

[0047] (6) Preparation of JTE-013@M1pep-M2MPs JTE-013@M2MPs was adjusted to 1 mg / mL and incubated with DSPE-PEG-M1pep at a mass ratio of 25:1 at 37°C for 4 hours, followed by overnight incubation at 4°C. The precipitate was collected after centrifugation at 14,000 rpm for 30 minutes the next day, and engineered extracellular vesicles targeting M1 macrophages were obtained and named JTE-013@M1pep-M2MPs.

[0048] Example 2: Physicochemical characterization and stability assessment of JTE-013@M1pep-M2MPs First, total protein content analysis showed that M2MPs carried a slightly higher protein content than MPs. Figure 1A); Flow cytometry analysis further revealed that the relative average fluorescence intensity of CD206 on the surface of M2MPs was significantly higher than that of the MPs group, indicating that it retained the surface marker characteristics of the maternal M2 macrophages (A). Figure 1 B).

[0049] Flow cytometry further examined the relative average fluorescence intensity of DID on the surface of M2MPs after modification with DSPE-PEG-M1pep at three mass ratios of 1:50, 1:25, and 1:12.5. The results showed that compared to the M2MPs group, the relative average fluorescence intensity of DID in M1pep-M2MPs (1:50), M1pep-M2MPs (1:25), and M1pep-M2MPs (1:12.5) was significantly increased. The relative average fluorescence intensity of DID in M1pep-M2MPs (1:25) and M1pep-M2MPs (1:12.5) was similar. Considering cost and material usage, 1:25 was selected as the optimal mass ratio for co-incubation. Figure 1 C). Furthermore, drug encapsulation efficiency testing showed no significant difference in encapsulation efficiency between M2MPs and M1pep-M2MPs for JTE-013, indicating that the surface modification of DSPE-PEG-M1pep does not affect the drug loading capacity of the final vesicles. Figure 1 D).

[0050] Subsequently, the particle size changes of M2MPs, M1pep-M2MPs, JTE-013@M2MPs, and JTE-013@M1pep-M2MPs at different time points were measured using nanoparticle tracking analysis (NTA) in PBS and PBS solution containing 10% FBS. The results showed that neither DSPE-PEG-M1pep modification nor JTE-013 loading significantly altered the colloidal stability of M2MPs. After 7 days of storage in PBS or PBS containing 10% FBS, the particle size of extracellular vesicles in all groups remained stable. Figure 1 E~F). The zeta potential stability of M2MPs, M1pep-M2MPs, JTE-013@M2MPs, and JTE-013@M1pep-M2MPs in PBS and PBS containing 10% FBS was evaluated at different time points using a zeta potential analyzer. The results showed that the modification of DSPE-PEG-M1pep and the loading of JTE-013 did not significantly affect the zeta potential of M2MPs. The zeta potentials of all extracellular vesicles remained stable during 7 days of storage in PBS or PBS containing 10% FBS. Figure 1 G~H).

[0051] JTE-013 working solutions containing 0 nM, 1 nM, 5 nM, 10 nM, 50 nM, 100 nM, 250 nM, 500 nM, and 1000 nM were prepared using complete culture medium. These solutions were then incubated with RAW264.7 cells induced with 500 ng / mL LPS for 24 hours. M1 polarization of RAW264.7 cells was assessed by flow cytometry. The results showed that with increasing JTE-013 concentration, the proportions of CD80-positive, CD86-positive, and CD80 and CD86-double-positive cells gradually decreased. Figure 1 I). Based on the reagent dosage, 1 nM was selected as the minimum effective concentration of free JTE-013.

[0052] Similarly, the potential and particle size distribution of M2MPs, M1pep-M2MPs, JTE-013@M2MPs, and JTE-013@M1pep-M2MPs were measured using a Zeta potential analyzer and NTA. The results showed that the Zeta potential of the extracellular vesicles in the above groups was stable at -10mV. Figure 2 A), with particle sizes uniformly distributed in the range of 100~1000 nm ( Figure 2 B). Furthermore, confocal fluorescence microscopy analysis of the distribution of Cy5-labeled M1pep showed that, compared to M2MPs, a significant Cy5 fluorescence signal was detected in M1pep-M2MPs, indicating that DSPE-PEG-M1pep had been successfully loaded onto the surface of M2MPs. Figure 2 C). Finally, transmission electron microscopy showed that M2MPs, M1pep-M2MPs, JTE-013@M2MPs, and JTE-013@M1pep-M2MPs all exhibited typical vesicle-like structures with clear membrane boundaries. After surface modification with DSPE-PEG-M1pep and drug loading with JTE-013, the overall morphology and membrane structure of the extracellular vesicles in each group did not show significant damage or aggregation. Figure 2 D).

[0053] Example 3: Evaluation of the targeting ability of M1pep-M2MPs in M1 macrophages and mice with acute pancreatitis To verify the targeting effect of M1pep-M2MPs on M1 macrophages, M2MPs or M1pep-M2MPs were co-incubated for 30 minutes with M2-polarized RAW264.7 cells (induced by 40 ng / mL IL-4 and 40 ng / mL IL-13 for 24 hours), M1-polarized RAW264.7 cells (induced by 500 ng / mL LPS for 24 hours), and untreated M0-state RAW264.7 cells. Flow cytometry and confocal microscopy were then used to evaluate the targeting ability of M1pep-M2MPs relative to M2MPs on M0, M1, and M2 macrophages. The results showed that M1pep-M2MPs exhibited significantly stronger targeting ability for M1 macrophages compared to M0 and M2 macrophages. Figure 2 E~G). Next, to evaluate the targeting effect of M1pep-M2MPs on acute pancreatitis tissue, a mouse model of acute pancreatitis was established by intraperitoneal injection of crotonin (50 μg / kg, once per hour, for a total of 7 times). M2MPs and M1pep-M2MPs stained with IR780 fluorescent dye were then injected via tail vein. The results showed that in the mouse model of acute pancreatitis, M1pep-M2MPs exhibited stronger fluorescence intensity in mouse pancreatic tissue compared to M2MPs, thus demonstrating good targeting ability of M1pep-M2MPs for pancreatic tissue in mice with acute pancreatitis. Figure 2 HI) Example 4: In vitro safety assessment of JTE-013@M1pep-M2MPs Whether treatment with JTE-013@M1pep-M2MPs caused cell damage was assessed using the Calcein-AM / PI double staining method. Specifically, immortalized pancreatic stellate cells, pancreatic acinar carcinoma cells, and bone marrow-derived macrophages were cultured with 0, 5, 15, and 50 μg / mL JTE-013@M1pep-M2MPs for 24, 48, and 72 hours, respectively. The staining results showed that only a very small number of PI-positive dead cells were observed in each group, while the majority were AM-positive live cells. Figure 3 A). Furthermore, the effect of JTE-013@M1pep-M2MPs on cell viability was evaluated using the CCK8 assay. Immortalized pancreatic stellate cells, pancreatic acinar carcinoma cells, and bone marrow-derived macrophages were cultured at concentrations of 0, 5, 15, and 50 μg / mL with their respective concentrations for 24, 48, and 72 hours. The results showed that under the above different concentrations and treatment times, the viability of all three cell types was not inhibited. Figure 3 (B~D). The above results indicate that JTE-013@M1pep-M2MPs have high biocompatibility and safety in vitro, and have no significant negative impact on cell viability.

[0054] Example 5: In vivo safety assessment of JTE-013@M1pep-M2MPs To assess the safety of JTE-013@M1pep-M2MPs in mice, 6-8 week old male C57BL / 6J mice were selected and acclimatized for one week in an SPF environment with a standard diet, with a weight of approximately 20 g. The mice were then randomly divided into 6 groups of 6 mice each. The specific experimental groups were as follows: (1) Control group: 100 μL of PBS solution and 100 μL of 10% DMSO + 40% PEG300 + 5% Tween-80 + 45% saline solution were injected once via the tail vein; (2) M2MPs group: 200 μL of PBS suspension containing 15 mg / kg M2MPs was injected once via the tail vein; (3) M1pep-M2MPs group: 200 μL of PBS suspension containing 15 mg / kg M1pep-M2MPs was injected once via the tail vein; (4) JTE-013 group: JTE-013 solution was prepared using a system of 10% DMSO + 40% PEG300 + 5% Tween-80 + 45% physiological saline, and 200 μL was injected once via the tail vein at a dose of 0.3 mg / kg; (5) JTE-013@M2MPs: 200 μL of PBS suspension containing 15 mg / kg JTE-013@M2MPs was injected once via the tail vein; (6) JTE-013@M1pep-M2MPs: A single injection of 200 μL of PBS suspension containing 15 mg / kg JTE-013@M1pep-M2MPs into the tail vein of mice.

[0055] Twenty-four hours after drug administration, serum samples were collected from mice, and the mice were euthanized. Tissue samples from the heart, liver, and other organs were then collected. Histological staining results showed that, compared to the Control group, the heart, liver, spleen, lung, kidney, and pancreas of the mice exhibited intact tissue structure with no obvious inflammatory infiltration or necrosis. Figure 4 A). Biochemical indicators from collected mouse serum samples also showed that serum amylase (Amylase, Figure 4 B) Lipase Figure 4 C) Alanine aminotransferase (ALT, Figure 4 D) Aspartate aminotransferase (AST), Figure 4 E), albumin (ALB, Figure 4 F), alkaline phosphatase (ALP, Figure 4 G), Blood urea nitrogen (BUN), Figure 4 H) and creatinine (CRE, Figure 4 I) All results were within the normal range and there were no significant differences between groups. These results indicate that JTE-013@M1pep-M2MPs at this dose showed no significant toxicity to the liver, kidneys, or pancreas of mice, demonstrating good safety.

[0056] Example 6: Evaluation of the effect of JTE-013@M1pep-M2MPs on RAW264.7 cell polarization Pancreatic acinar cancer cells were treated in vitro with cholecystokinin octapeptide for 12 hours to induce cell damage and release of S1P. Conditional supernatant (CM) enriched with natural S1P from this process was then prepared from the resulting CM. CCK ). In CM CCK Alternatively, under conditions of exogenous S1P addition, six intervention groups were simultaneously set up, including PBS, M2MPs, M1pep-M2MPs, free JTE-013, JTE-013@M2MPs, and JTE-013@M1pep-M2MPs, to evaluate the effects of different treatments, such as JTE-013@M1pep-M2MPs, on M1 polarization in RAW264.7 cells. Figure 5 A), the specific groups are as follows: CM CCK + PBS: in CM CCK Under the conditions, PBS solution was added as a control, and the mixture was incubated for 24 hours. CM CCK + M2MPs: In CM CCK Under these conditions, add M2MPs to a final concentration of 50 nM and incubate for 24 hours; CM CCK + M1pep-M2MPs: In CM CCK Under these conditions, add M1pep-M2MPs to a final concentration of 50 nM and incubate for 24 hours; CM CCK + JTE-013: In CM CCK Under these conditions, add JTE-013 to a final concentration of 1 nM and incubate for 24 hours; CM CCK + JTE-013@M2MPs: In CM CCK Under these conditions, JTE-013@M2MPs with a final concentration of 50 nM were added and cultured for 24 hours. CM CCK + JTE-013@M1pep-M2MPs: In CM CCKUnder these conditions, JTE-013@M1pep-M2MPs with a final concentration of 50 nM were added and cultured for 24 hours.

[0057] The grouping under the condition of adding S1P from the outside is the same as above, the difference being that CM is added. CCK Replace with a complete culture medium containing 1 μM S1P.

[0058] After 24 hours of culture, flow cytometry was used to detect macrophage M1 polarization-related indices to evaluate the regulatory effect of JTE-013@M1pep-M2MPs on S1P-induced macrophage M1 polarization. Results showed that in CM... CCK Under the treatment conditions, compared with the CMCCK + PBS group, CMCCK + M2MPs group, CMCCK + M1pep-M2MPs group, CMCCK + JTE-013 group, and CMCCK + JTE-013@M2MPs group, the MHC II concentration in the JTE-013@M1pep-M2MPs group was significantly lower. + Cell ratio ( Figure 5 B) and CD80 + cell( Figure 5 C) The proportion was significantly reduced. Under the condition of exogenous S1P addition, compared with other treatment groups, the MHC II concentration in the JTE-013@M1pep-M2MPs group was significantly reduced. + Cell ratio ( Figure 5 D) and CD80 + cell( Figure 5 The proportion of CD86 (E) was also significantly reduced. qRT-PCR results showed that, compared to other treatment groups, the JTE-013@M1pep-M2MPs group had a significantly lower proportion of CD86 (E). Figure 5 F) and TNF-α Figure 5 The expression of G was significantly reduced, and MRC1 ( Figure 5 H) and Arg1 ( Figure 5 The expression of I) was significantly increased.

[0059] To evaluate which signaling pathways JTE-013@M1pep-M2MPs mediates to inhibit M1 polarization in RAW264.7 cells, Western blotting experiments were performed on the phosphorylation levels of several proteins. Figure 5 J). The results showed that, compared with PBS treatment alone, using CM CCKCulture or exogenous S1P significantly increased the relative expression levels of p-PI3K, p-P38, p-JNK, p-ERK, and NLRP3 proteins in RAW264.7 cells. Compared to exogenous S1P treatment, the relative expression levels of p-PI3K, p-P38, p-JNK, p-ERK, and NLRP3 proteins in RAW264.7 cells were decreased in the S1P + M2MPs, S1P + M1pep-M2MPs, S1P + JTE-013, S1P + JTE-013@M2MPs, and S1P + JTE-013@M1pep-M2MPs groups. Among these, JTE-013@M1pep-M2MPs treatment significantly reversed the S1P-induced reduction in p-PI3K protein expression in RAW264.7 cells. Figure 5 K), p-P38 protein ( Figure 5 L), p-JNK protein ( Figure 5 M), p-ERK protein ( Figure 5 N) and upregulation of NLRP3 protein expression levels ( Figure 5 O). The above results indicate that JTE-013@M1pep-M2MPs may inhibit macrophage M1 polarization by mediating the blocking of the PI3K-AKT pathway, the MAPK pathway, and the inflammasome pathway.

[0060] Example 7: JTE-013@M1pep-M2MPs significantly improved the state of acute pancreatitis induced by taeniacin in mice. Male C57BL / 6J mice, aged 6-8 weeks and weighing approximately 20 g, were selected as research subjects. They were acclimatized to a standard diet in an SPF environment for one week. Before the experiment, the mice were fasted for 12 hours. After fasting, they were injected intraperitoneally with 50 μg / kg of rain frog, once every hour for a total of 7 times, to establish a mouse model of acute pancreatitis injury. The mice were randomly divided into 7 groups of 6 mice each. At 2 and 3 hours after model establishment, PBS, M2MPs, M1pep-M2MPs, JTE-013, JTE-013@M2MPs, and JTE-013@M1pep-M2MPs were injected intravenously via the tail vein, respectively. The mice were euthanized 24 hours after model establishment, and their pancreas, lung tissue, and serum were collected. Figure 6 A). The specific experimental groups are as follows: (1) Control group: In normal mice, at 8 hours and 9 hours after the start of the experiment, 100 μL of PBS solution and 100 μL of 10% DMSO + 40% PEG300 + 5% Tween-80 + 45% saline solution were injected once via the tail vein. (2) AP + PBS group: 200 μL of PBS was injected once via tail vein at 2 hours and 3 hours after modeling was completed; (3) AP + M2MPs group: At 2 hours and 3 hours after modeling, 200 μL of PBS suspension containing 15 mg / kg M2MPs was injected once via tail vein. (4) AP + M1pep-M2MPs group: At the 2nd and 3rd hour after modeling, 200 μL of PBS suspension containing 15 mg / kg M1pep-M2MPs was injected once via the tail vein; (5) AP + JTE-013 group: At 2 hours and 3 hours after modeling, JTE was prepared by single injection via tail vein using a system of 10% DMSO + 40% PEG300 + 5% Tween-80 + 45% saline. 013 solution, administered via a single tail vein injection of 200 μL at a dose of 0.3 mg / kg; (6) AP + JTE-013@M2MPs group: At the 2nd and 3rd hour after modeling, 200 μL of PBS suspension containing 15 mg / kg JTE-013@M2MPs was injected once via the tail vein; (7) AP + JTE-013@M1pep-M2MPs group: At the 2nd and 3rd hour after modeling, mice were injected once via the tail vein with 200 μL of PBS suspension containing 15 mg / kg JTE-013@M1pep-M2MPs.

[0061] Histochemical staining results showed that, compared with the Control group, the pancreatic tissue of mice in the AP + PBS group exhibited severe edema, extensive inflammatory cell infiltration, and tissue necrosis, indicating that an acute pancreatitis model was successfully established. Compared with the AP + PBS group, the edema, inflammatory infiltration, and tissue necrosis were all reduced in the AP + M2MPs, AP + M1pep-M2MPs, AP + JTE-013, AP + JTE-013@M2MPs, and AP + JTE-013@M1pep-M2MPs groups. However, the JTE-013@M1pep-M2MPs group showed the most significant improvement in pancreatic edema, inflammatory cell infiltration, and tissue necrosis, as well as the most significant improvement in pulmonary hemorrhage, septal thickening, and inflammatory infiltration. Figure 6 B~C), the corresponding pancreatic histopathological scores also indicate that JTE-013@M1pep-M2MPs can effectively improve pancreatic edema, inflammatory cell infiltration and tissue necrosis (B~C). Figure 6F), which also has a significant ameliorative effect on pancreatitis-related lung injury, and can effectively alleviate pulmonary hemorrhage, alveolar septal thickening and inflammatory cell infiltration (F). Figure 6 G). Among them, JTE-013@M1pep-M2MPs can reduce the pancreatic wet weight / body weight ratio, further demonstrating its effectiveness in reducing pancreatic edema. Figure 6 H).

[0062] Meanwhile, staining for CD45, an immune cell infiltration marker, and MPO, a neutrophil activation marker, showed that compared to the Control group, the expression of CD45 and MPO in the pancreatic tissue of mice in the AP + PBS group was significantly increased. Compared to the AP + PBS group, the expression of CD45 and MPO was decreased in the AP + M2MPs, AP + M1pep-M2MPs, AP + JTE-013, AP + JTE-013@M2MPs, and AP + JTE-013@M1pep-M2MPs groups, with the most significant decrease in CD45 and MPO expression in the JTE-013@M1pep-M2MPs group. Figure 6 D~E). ELISA test results ( Figure 6 I~J) and quantitative analysis of staining results ( Figure 6 K~L) also showed that after treatment with JTE-013@M1pep-M2MPs, the infiltration of immune cells and activation of neutrophils in pancreatic tissue were significantly reduced.

[0063] The effects of different treatments on pancreatic injury and systemic inflammation in mice were assessed by ELISA detection of relevant inflammatory markers in collected mouse serum. Results showed that, compared to the Control group, the expression of TNF-α, IL-1β, and IL-6 in the pancreatic tissue of mice in the AP + PBS group was significantly increased. Compared to the AP + PBS group, the expression of TNF-α, IL-1β, and IL-6 was decreased in the AP + M2MPs, AP + M1pep-M2MPs, AP + JTE-013, AP + JTE-013@M2MPs, and AP + JTE-013@M1pep-M2MPs groups. The JTE-013@M1pep-M2MPs group showed the most significant decrease in the expression of TNF-α, IL-1β, and IL-6. Figure 6 M~O). That is, treatment with JTE-013@M1pep-M2MPs significantly reduced the levels of amylase and lipase in serum.

[0064] Example 8: JTE-013@M1pep-M2MPs reprogramming macrophages in acute pancreatitis tissue The pancreatic tissues collected in Example 7 were numbered, and the correspondence is shown below: (1) Group G1 is the Control group; (2) Group G2 was the AP + PBS group; (3) Group G3 is the AP + M2MPs group; (4) Group G4 is the AP + M1pep-M2MPs group; (5) Group G5 is AP + JTE-013 group; (6) Group G6 is AP + JTE-013@M2MPs group; (7) Group G7 is AP + JTE-013@M1pep-M2MPs group.

[0065] Immunofluorescence staining was performed on the pancreatic tissues. F4 / 80 was used to label macrophages in the pancreatic tissue, CD86 was used to label M1 macrophages, and CD206 was used to label M2 macrophages. The results showed that, compared to group G1, the fluorescence intensity of F4 / 80 + CD86 was significantly enhanced in group G2. Compared to group G2, the fluorescence intensity of F4 / 80 + CD86 was weakened in groups G3, G4, G5, G6, and G7. In contrast, almost no CD86 fluorescence was observed in group G7. This indicates that treatment with JTE-013@M1pep-M2MPs significantly reduced the number of M1 macrophages in the pancreatic tissue, alleviating tissue damage and systemic inflammatory response in acute pancreatitis. Figure 7 A and Figure 7 E). Meanwhile, compared to other treatment groups, the fluorescence intensity of F4 / 80 + CD206 in the JTE-013@M1pep-M2MPs group was significantly enhanced, indicating that JTE-013@M1pep-M2MPs treatment can significantly increase the proportion of M2 macrophages (E). Figure 7 B and Figure 7 F). The above results indicate that JTE-013@M1pep-M2MPs can effectively regulate macrophage polarization and promote the transformation of inflammation into a repair phenotype.

[0066] Furthermore, immunohistochemical results showed that, compared to the G1 group, the phosphorylation level of NF-κB in the pancreatic tissue of mice in the G2 group was significantly increased, and the nuclear translocation of phosphorylated NF-κB was significantly enhanced. Simultaneously, the expression level of SPHK1 protein was also significantly upregulated. Compared to the G2 group, the phosphorylation level of NF-κB and the expression level of SPHK1 protein were decreased in the G3, G4, G5, G6, and G7 groups. In contrast, the decrease in NF-κB phosphorylation level and SPHK1 protein expression level was most significant in the G7 group. Figure 7 C~D, Figure 7 G~H), and the content of S1P in its serum was also significantly reduced ( Figure 7 I).

[0067] The protein expression levels of inflammatory factors such as TNF-α, IL-1β, and IL-6 in mouse pancreatic tissue were detected and quantified using Western blotting. The results showed that JTE-013@M1pep-M2MPs significantly reduced the production of IL-1β, IL-6, and TNF-α in pancreatic tissue, and inhibited NF-κB phosphorylation and SPHK1 expression. Figure 7 The above results indicate that JTE-013@M1pep-M2MPs can reprogram M1 macrophages in acute pancreatitis tissue, reshape the immune environment of pancreatic tissue, block the activation of the S1P-S1PR2 signaling pathway, reduce the production of pro-inflammatory factors, improve pancreatic acinar cell damage, and thus improve pancreatic injury and systemic inflammatory response.

[0068] Example 9: Transcriptome sequencing reveals the molecular signaling pathway by which JTE-013@M1pep-M2MPs alleviate pancreatic injury in acute pancreatitis. To more comprehensively evaluate the effects of JTE-013@M1pep-M2MPs on tissue damage in acute pancreatitis, transcriptome sequencing was used to analyze pancreatic tissue from acute pancreatitis model mice (AP group) and mice treated with JTE-013@M1pep-M2MPs (AP_MPs group). Figure 8 A). Correlation plots and PCA cluster analysis showed that JTE-013@M1pep-M2MPs treatment significantly affected the pancreatic tissue of mice with acute pancreatitis and produced significant differentiation from mice with acute pancreatitis model (A). Figure 8 B~C). Volcano plots and differential heatmaps showed that, compared to the AP group, the AP_MPs group had 693 significantly upregulated genes and 787 significantly downregulated genes (8D~E). Among these differentially expressed genes, the AP_MPs group showed significantly downregulated expression of inflammatory genes and significantly upregulated expression of normal pancreatic secretory genes. Figure 8 F). KEGG enrichment analysis showed that inflammatory signaling pathways such as IL-17, TNF-α, and NF-κB were significantly enriched in the AP_MPs group, and the pancreatic secretion and protein digestion and absorption pathways of normal pancreatic function were also significantly enriched, indicating that JTE-013@M1pep-M2MPs has the effect of inhibiting inflammation in acute pancreatitis tissue and restoring normal pancreatic function. Figure 8 G). GO enrichment analysis showed differential alterations in the S1P signaling pathway, and differential enrichment was also observed in macrophage activation, inflammatory secretion, and normal pancreatic secretion. Figure 8 H). GSEA enrichment also indicated that JTE-013@M1pep-M2MPs effectively inhibited the overactivation of key inflammation-related pathways in pancreatic tissue during acute pancreatitis and promoted the recovery of pancreatic exocrine function. Figure 8 I).

[0069] To further validate the transcriptome analysis results, the expression levels of inflammatory markers in the pancreatic tissues of mice in different treatment groups were analyzed by qRT-PCR. The results showed that JTE-013@M1pep-M2MPs could significantly reduce the expression of the inflammatory marker Sphk1 (…). Figure 8 J), Cxcl2 ( Figure 8 K), MCP1 ( Figure 8 L), CD86 ( Figure 8 M), iNOS ( Figure 8 The expression of N).

[0070] In the description of this specification, the references to terms such as "one embodiment / mode," "some embodiments / modes," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment / mode or example is included in at least one embodiment / mode or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment / mode or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments / modes or examples. Furthermore, without contradiction, those skilled in the art can combine and integrate the different embodiments / modes or examples described in this specification, as well as the features of different embodiments / modes or examples.

[0071] It should be noted that in this invention, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element. In this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise expressly specified.

[0072] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features of the invention herein.

Claims

1. An engineered extracellular vesicle, characterized in that, The engineered extracellular vesicles are derived from M2 macrophages, with an M1pep homing-targeting peptide coupled to their surface and an S1PR2 inhibitor loaded inside. The amino acid sequence of the M1pep homing-targeting peptide is shown in SEQ ID NO:1, and the S1PR2 inhibitor is JTE-013. The M1pep homing-targeting peptide is coupled to the surface of the extracellular vesicles via a DSPE-PEG-Mal linker.

2. A method for preparing engineered extracellular vesicles as described in claim 1, characterized in that, Includes the following steps: Provides extracellular vesicles derived from M2 macrophages; The S1PR2 inhibitor was co-incubated with extracellular vesicles derived from the M2 macrophages to obtain extracellular vesicles loaded with the S1PR2 inhibitor. The M1pep homing-targeting peptide was reacted with DSPE-PEG-Mal linker to obtain DSPE-PEG-M1pep; The engineered extracellular vesicles were obtained by co-incubating the DSPE-PEG-M1pep with the extracellular vesicles loaded with the S1PR2 inhibitor.

3. The method for preparing engineered extracellular vesicles according to claim 2, characterized in that, The provision of M2 macrophage-derived extracellular vesicles includes: Macrophage M2 polarization was induced by anti-inflammatory cytokines, and cell culture supernatant was collected. Extracellular vesicles derived from the M2 macrophages were collected from the cell culture supernatant by differential centrifugation.

4. The method for preparing engineered extracellular vesicles according to claim 2, characterized in that, The S1PR2 inhibitor is JTE-013; the extracellular vesicles loaded with the S1PR2 inhibitor are extracellular vesicles loaded with JTE-013; the concentration of JTE-013 is 50~150 μg / mL.

5. The method for preparing engineered extracellular vesicles according to claim 2, characterized in that, The concentration of extracellular vesicles derived from M2 macrophages is 0.5–2 mg / mL.

6. The method for preparing engineered extracellular vesicles according to claim 4, characterized in that, The mass ratio of the co-incubation of the DSPE-PEG-M1pep and the extracellular vesicles loaded with JTE-013 is 1:(10~50).

7. The use of the engineered extracellular vesicles according to claim 1 in the preparation of a medicament for treating acute pancreatitis.

8. The application according to claim 7, characterized in that, The drug comprises the engineered extracellular vesicles and their pharmaceutically acceptable carriers.

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