Iridium nanozyme-loaded endotoxin-tolerant dendritic cell-derived biomimetic exosomes, preparation method, and application thereof

Abstract

The present invention belongs to the field of biopharmaceuticals, and specifically relates to a bionic exosome derived from endotoxin-tolerant dendritic cells loaded with iridium nanozymes, a preparation method and an application thereof. The present invention provides an exosome mimetic (ETDC-EM) derived from endotoxin-tolerant dendritic cells, and Ir NPs-PVP is loaded into ETDC-EM. In vitro experiments have shown that ETDC-EM has good anti-inflammatory effects, and Ir NPs-PVP has good antioxidant effects. In vivo experiments have shown that ETDC-EM / Ir NPs-PVP still has good anti-inflammatory and antioxidant effects in ALF model mice, and ETDC-EM and Ir NPs-PVP have synergistic therapeutic effects.

Description

Technical Field

[0001] The present invention belongs to the field of biopharmaceuticals, and specifically relates to a bionic exosome derived from endotoxin-tolerant dendritic cells loaded with iridium nanozymes, a preparation method and an application thereof. Background Art

[0002] Acute liver failure refers to a group of clinical syndromes caused by multiple reasons such as drug poisoning, liver ischemia, viral hepatitis, autoimmune hepatitis, etc., in which a large number of immune cells infiltrate the liver, produce inflammatory cytokines, and cause acute necrosis of a large number of hepatocytes and liver function damage, resulting in multiple organ dysfunctions such as jaundice, coagulopathy, and hepatic encephalopathy. Due to oxidative stress, the mitochondrial and endoplasmic reticulum functions in ALF patients are impaired, which can lead to liver cell death, release inflammatory mediators, cause inflammatory reactions and cell apoptosis, and further lead to the occurrence of acute liver failure. Therefore, anti-inflammatory and antioxidant treatment are extremely critical in the treatment of ALF. Most ALF patients are treated with symptomatic supportive treatment in clinical practice, but some conventional drugs are limited in clinical treatment due to poor solubility, nonspecific organ toxicity, and poor blood circulation stability in the treatment of ALF. In addition, ALF can be treated by orthotopic liver transplantation, but its clinical application is limited by the scarcity of liver donors, high cost, and organ transplant rejection. Therefore, it is urgent to find a new, effective and safe treatment method to provide a new way for the treatment of ALF.

[0003] Dendritic cells (DC) are a unique type of immune cell that serves as a bridge between the innate and acquired immune systems. DCs originate from hematopoietic stem cells and are important antigen-presenting cells in the body. They can aggregate to sites of infection or infiltrate pathological tissues to search for antigens that can be used to activate effector cells. DCs are essential for initiating cellular and humoral immune responses and protecting the body from infection and clearing tumor cells. DCs can also use special surface receptors such as endocytic receptors or C-type lectin receptors to absorb antigens. In addition, DCs are equipped with a series of extracellular and intracellular pattern recognition receptors to sense different danger signals. After sensing the danger signal, DCs are activated, upregulate co-stimulatory molecules, produce various cytokines and chemokines, absorb and process antigens, and then migrate to the lymph nodes, where they transport antigens to CD8 + T cells and CD4 + T cells present antigens.

[0004] Endotoxin tolerance refers to the weakened or even no response of an organism or cell to a lethal dose of lipopolysaccharide after being pre-exposed to a low dose of LPS. In the state of endotoxin tolerance, the body can tolerate the release of lethal doses of endotoxins and cytokines and the reperfusion injury of cells or tissues caused by inflammatory responses. The main characteristics of endotoxin tolerance are the downregulation of inflammatory mediators such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) and the upregulation of anti-inflammatory cytokines such as IL-10 and transforming growth factor (TGF-β), and the cell phenotype changes from a pro-inflammatory response to an anti-inflammatory response. Therefore, endotoxin tolerance is often considered to be a self-protection mechanism of the host against excessive inflammatory responses in the body.

[0005] Extracellular vesicles (EVs) refer to membrane-enclosed vesicles produced and secreted by cells in organisms. According to the different biogenesis and secretion modes of EVs, they can be divided into apoptotic bodies, microvesicles and exosomes (Exo). Once released outside the cell, extracellular vesicles can reach target cells for intercellular communication. A large number of studies have shown that EVs can regulate immune function and reduce tissue damage. However, due to the low yield of extracellular vesicles and the cumbersome acquisition process, it has always been an obstacle to its widespread use in clinical practice. Exosome mimics (EM), as an alternative to exosomes, solve the problem of low exosome yield and maintain the excellent properties of exosomes. Summary of the invention

[0006] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide a bionic exosome derived from endotoxin-tolerant dendritic cells loaded with iridium nanozymes, a preparation method and an application thereof.

[0007] In a first aspect of the present invention, there is provided a bionic exosome derived from endotoxin-tolerant dendritic cells loaded with iridium nanozyme, which comprises bionic exosomes derived from endotoxin-tolerant dendritic cells and iridium nanozyme particles, wherein the iridium nanozyme particles are embedded in the bionic exosomes.

[0008] The second aspect of the present invention provides a method for preparing the bionic exosomes derived from endotoxin-tolerant dendritic cells loaded with iridium nanozymes as described above, comprising the following steps:

[0009] (1) Preparation of endotoxin-tolerant dendritic cells;

[0010] (2) Preparation of iridium nanozyme particles by ethanol thermal reduction method;

[0011] (3) The endotoxin-tolerant dendritic cells prepared in step (1) are resuspended with the dispersion prepared with the iridium nanozyme particles prepared in step (2), and the resuspended liquid is passed through a 1 μm polycarbonate filter membrane using a liposome extruder. The extruded sample is centrifuged at 20,000 g for 30 min, the supernatant is removed, and the precipitate is collected to obtain biomimetic exosomes derived from endotoxin-tolerant dendritic cells loaded with iridium nanozyme.

[0012] Preferably, step (1) comprises the following steps:

[0013] Dendritic cells were added to RPMI-1640 complete medium containing GM-CSF and IL-4 cytokines, seeded in well plates, and cultured in a 37°C constant temperature incubator;

[0014] On the 4th day of culture, the culture supernatant in the well plate was aspirated, and RPMI-1640 complete medium containing GM-CSF and IL-4 cytokines was added again;

[0015] On the 5th day of culture, 100 ng / mL LPS was added for stimulation for 48 h. On the 7th day of culture, the culture supernatant in the well plate was aspirated, and fresh RPMI-1640 complete medium containing GM-CSF and IL-4 cytokines was added, and 1 μg / mL LPS was added for stimulation for 24 h to obtain endotoxin-tolerant dendritic cells.

[0016] Preferably, the dendritic cells are dendritic cells derived from mouse bone marrow.

[0017] Preferably, step (2) comprises the following steps:

[0018] IrCl 3 ·3H 2 O was dissolved in pure water and sonicated in an ultrasonicator for more than 3 h to obtain a transparent light yellow solution;

[0019] Then, PVP was dissolved in ethanol solution and mixed evenly. Finally, IrCl 3 ·3H 2 The O aqueous solution was slowly dropped into the PVP-ethanol solution, and the mixture was stirred at room temperature overnight;

[0020] The next day, the mixture was condensed and refluxed at 100° C. for 6-8 h to obtain a black solution, and then excess solvent was removed by a rotary evaporator to obtain a black product, which was then dried using a vacuum freeze dryer to obtain iridium nanozyme particles.

[0021] Preferably, step (3) comprises the following steps: 6Each cell was taken as one unit and resuspended in 1 mL of dispersion solution. The amount of iridium nanozyme particles in the dispersion solution was 2 mg / mL.

[0022] The third aspect of the present invention provides the use of the bionic exosomes derived from endotoxin-tolerant dendritic cells loaded with iridium nanozymes as described above for preparing a drug for treating acute liver failure.

[0023] In a fourth aspect, the present invention provides a drug for treating acute liver failure, comprising the bionic exosomes derived from endotoxin-tolerant dendritic cells loaded with iridium nanozymes as described above.

[0024] Preferably, the drug is an injectable preparation.

[0025] The present invention uses an effective extrusion method to obtain exosome mimics EMs from ETDC. Compared with the exosomes secreted by ETDC, the EM yield is greatly improved, and on the basis of retaining the biologically active molecules of the exosomes, the proportion of vesicles positive for its specific marker CD63 is significantly increased. Polyvinylpyrrolidone (PVP) stabilized iridium nanoparticles (Ir NPs-PVP) are prepared by a one-pot method. Ir NPs-PVP has good biocompatibility and dispersibility, a simple and convenient synthesis process, and a high yield. Then, Ir NPs-PVP and ETDC are extruded simultaneously through a polycarbonate membrane by an extrusion method to construct an ETDC-EM / Ir NPs-PVP complex.

[0026] Ir NPs-PVP has multi-enzyme mimicking activity and can effectively eliminate ROS. In the anti-inflammatory experiment of RAW264.7 cells, ETDC-EM can effectively reduce inflammation and play a good role in cells. At the same time, as a carrier of Ir NPs-PVP, ETDC-EM can not only play a good anti-inflammatory effect in vivo, but also prolong the circulation time of Ir NPs-PVP in vivo and delay the antioxidant effect of Ir NPs-PVP. Therefore, the ETDC-EM / Ir NPs-PVP complex of the present invention can play an anti-inflammatory and antioxidant role at the same time, and can play a synergistic effect, making the therapeutic effect more effective.

[0027] Specifically, ETDC-EM / Ir NPs-PVP was injected into the tail vein, and macroscopic observation showed that the congestion of liver tissue in ALF mice treated with ETDC-EM / Ir NPs-PVP was significantly reduced. Hematoxylin and eosin (HE) staining of liver tissue confirmed that ETDC-EM / Ir NPs-PVP alleviated the symptoms of liver edema degeneration, tissue damage, and inflammatory cell infiltration in ALF mice. The concentrations of inflammatory factors including interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and serum alanine aminotransferase and aspartate aminotransferase in mice treated with ETDC-EM / Ir NPs-PVP decreased significantly. In addition, mitochondria in mouse liver tissue are important organelles for ROS production and clearance. Mitochondrial dysfunction plays a vital role in oxidative stress diseases, and oxidative stress of the endoplasmic reticulum can also induce ROS release. In summary, ETDC-EM / Ir NPs-PVP exhibits excellent anti-inflammatory and antioxidant effects and has clinical translation potential in alleviating ALF.

[0028] In summary, the present invention provides an endotoxin-tolerant dendritic cell-derived exosome mimetic (ETDC-EM), and Ir NPs-PVP is loaded into ETDC-EM. In vitro experiments have shown that ETDC-EM has good anti-inflammatory effects, and IrNPs-PVP has good antioxidant effects. In vivo experiments in ALF model mice have verified that ETDC-EM / Ir NPs-PVP still has good anti-inflammatory and antioxidant effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, without paying creative labor, other drawings obtained based on these drawings still belong to the scope of the present invention.

[0030] Figure 1In the figure, (a) physical image of Ir NPs-PVP; TEM images of Ir NPs-PVP (b), ETDC-EM (c) and ETDC-EM / IrNPs-PVP (d); (e) particle size statistics of Ir NPs-PVP; (f) particle size images of ETDC-EM and ETDC-EM / Ir NPs-PVP; (g) ζ potential of Ir NPs-PVP, ETDC-EM and ETDC-EM / Ir NPs-PVP; (h) UV absorption spectra of Ir NPs-PVP, ETDC-EM and ETDC-EM / Ir NPs-PVP; (i) X-ray photoelectron spectroscopy (XPS) of Ir NPs-PVP;

[0031] Figure 2 (a). Ir NPs-PVP on H 2 O 2 / UV system to scavenge -OH; (b) Effect of different concentrations of IrNPs-PVP on H 2 O 2 (c) The same concentration of Ir NPs-PVP has an effect on the clearance of H 2 O 2 (d) CAT enzyme activity of Ir NPs-PVP at different concentrations; (e) POD enzyme activity under different concentrations of Ir NPs-PVP; (f) Flow cytometry verification of the ROS scavenging effect of Ir NPs-PVP;

[0032] Figure 3 In the figure, (a). ELISA detection of TNF-α in the culture supernatant of RAW264.7 cells; (b). ELISA detection of IL-1β in the culture supernatant of RAW264.7 cells; (c). Flow cytometry detection of the expression level of CD86 after co-culture of RAW264.7 and ETDC-EM;

[0033] Figure 4 In the figure, (a). ICP determination of Ir NPs-PVP in ETDC-EM; (b) UV-visible spectrum of ETDC-EM / Ir NPs-PVP; (c) ICP detection of the metabolism of Ir NPs-PVP in ETDC-EM / Ir NPs-PVP; (d) Fluorescence image of mouse liver taken by in vivo imaging system; (e) In vivo quantitative imaging of liver;

[0034] Figure 5(a). Hemolysis test 200ug / ml+EM, 500ug / ml+EM, 1mg / ml+EM, 2mg / ml+EM; (b). Quantitative results of Figure (a); (c). Detection of cell activity after CCK8 RAW264.7 cultured with ETDC-EM / Ir NPs-PVP; (d) HE staining of major organs (kidney, lung, spleen, liver, heart);

[0035] Figure 6 (a) Physical images of mouse livers in the healthy group, ALF group, and after treatment with Ir NPs-PVP, ETDC-EM, and ETDV-EM / Ir NPs-PVP; (b) HE staining of the livers of mice in each group; (c) Serum ALT levels of mice in each group; (d) Serum AST levels of mice in each group;

[0036] Figure 7 (a) Immunohistochemical staining of IL-1β in liver tissue of each group of mice; (b) Immunohistochemical staining of TNF-α in liver tissue of each group of mice; (c) TNF-α immunohistochemical quantification; (d) Immunohistochemical quantification of IL-1β; (e) Expression of TNF-α in serum of each group of mice; (f) Expression of IL-1β in serum of each group of mice; (g) Immunofluorescence analysis of ROS expression in liver tissue of mice after different treatments. DETAILED DESCRIPTION

[0037] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings.

[0038] The experimental materials used in the following examples are as follows.

[0039] 1. Experimental Animals

[0040] Healthy clean grade male C57 BL / 6 mice 6-8 weeks old. The Animal Ethics Committee of the Experimental Animal Center of the First Hospital of Wenzhou Medical University approved the protocols for all animal studies conducted in this study. All animals were treated in accordance with the guidelines in the Guide for the Care and Use of Laboratory Animals. All C57BL / 6 were purchased from Zhejiang Life River Laboratory Animal Technology Co., Ltd.

[0041] 2. Experimental Reagents

[0042]

[0043]

[0044]

[0045]

[0046] 3. Preparation of main reagents

[0047] ① RPMI-1640 complete medium

[0048] Add 45 mL of RPMI-1640 medium to a 50 mL centrifuge tube, then add 5 mL of inactivated fetal bovine serum again, and finally add 500 ul of penicillin-streptomycin mixture (streptomycin 100 μg / mL, penicillin 100 U / mL) to the above medium, mix well, and finally seal with sealing film and store aseptically in a 4°C refrigerator for later use.

[0049] ②RPMI-1640 complete medium containing GM-CSF and IL-4 cytokines

[0050] Add 200ul of 20ng / mL GM-CSF and 50ul of 5ng / mL IL-4 to the above RPMI-1640 complete medium and mix well to avoid affecting the experimental results. The medium containing cytokines needs to be prepared and used immediately.

[0051] ③1mg / mL LPS

[0052] Weigh 10 mg of LPS powder, dissolve it in 10 mL of sterile PBS, mix well and dispense into enzyme-free sterile 1.5 mL eppendorf tubes, and store at -20°C for later use.

[0053] ④Phosphate buffer

[0054] Dissolve 120 g of PBS powder in 2 L of pure water, put it into a glass bottle, sterilize it at high temperature and store it in a refrigerator at 4 °C for later use.

[0055] Example 1 Preparation of ETDC-EM / Ir NPs-PVP:

[0056] 1. Extraction and Culture of Mouse Bone Marrow-derived Dendritic Cells

[0057] Male C57BL / 6 mice aged 6-8 weeks and weighing about 25g were killed by cervical dislocation. The tibia and femur were removed, and the muscles and skin on the bones were carefully removed to avoid contamination caused by bone breakage. The clean leg bones were soaked in 75% alcohol for 5 min and transferred to the clean bench. The leg bones were washed twice in sterile PBS, and then the femurs and tibias were cut off respectively and rinsed with sterile PBS to obtain a cell suspension. The cell suspension was filtered using a 200-mesh copper mesh and then transferred to a clean 15mL centrifuge tube. After centrifugation at 450g for 5 min, the supernatant was removed to collect the cell pellet; the red blood cell lysis buffer was added to resuspend the cell pellet, and the cell pellet was placed in a 4℃ refrigerator for 10 min after being blown evenly; an equal volume of PBS was added to terminate the lysis, and the cell pellet was centrifuged at 450g for 5 min again; the supernatant was discarded to obtain bone marrow-derived dendritic cells (BMDC). BMDCs were added to RPMI-1640 complete medium containing GM-CSF and IL-4 cytokines, seeded in 6-well plates, and cultured in a 37°C constant temperature incubator.

[0058] On the 4th day of culture, the culture supernatant in the well plate was removed by aspiration, and RPMI-1640 complete medium containing GM-CSF and IL-4 cytokines was added again.

[0059] On the 5th day of culture, a small dose (100 ng / mL) of LPS was added for stimulation for 48 h. On the 7th day of culture, the culture supernatant in the well plate was aspirated, and fresh RPMI-1640 complete medium containing GM-CSF and IL-4 cytokines was added. A large dose (1 μg / mL) of LPS was added for stimulation for 24 h to obtain endotoxin-tolerant DC (ETDC).

[0060] On the fifth day of culture, the culture supernatant in the well plate was aspirated, and fresh RPMI-1640 complete medium containing GM-CSF and IL-4 cytokines was added without adding LPS; on the seventh day of culture, the culture supernatant in the well plate was aspirated, and fresh RPMI-1640 complete medium containing GM-CSF and IL-4 cytokines was added, and a large dose (1 μg / mL) of LPS was added to stimulate for 24 hours to obtain mature DCs (mDCs).

[0061] The cell morphology of BMDC, mDC and ETDC was observed by optical microscopy at different time points.

[0062] The bone marrow-derived dendritic cells (BMDC) prepared by the above method were analyzed by flow cytometry and confirmed that the positive rate of cells expressing DC marker CD11 was 66.1% ( Figure 1-1a). BMDCs were stimulated with a high dose of lipopolysaccharide (LPS) to become mature DCs (mDCs). After BMDCs were stimulated with a low dose of LPS, they were stimulated with a high dose of LPS again to obtain endotoxin-tolerant DCs (ETDCs). Compared with mDCs, optical microscopy results showed that ETDCs after two LPS stimulations were similar in morphology to BMDCs without LPS stimulation, while mDCs that were stimulated with only one high dose of LPS extended more obvious and more protrusions ( Figure 1-1 b).

[0063] 2. Preparation of Iridium Nanozymes

[0064] Iridium nanozyme particles with good dispersibility were prepared by ethanol thermal reduction method. First, IrCl 3 ·3H 2 O (2.96 mg) was dissolved in 4 mL pure water and sonicated for more than 3 h in an ultrasonic instrument to obtain a transparent light yellow solution. Then, PVP (18.6 mg) was dissolved in 4 mL ethanol solution and mixed well. Finally, IrCl 3 ·3H 2 O aqueous solution was slowly dripped into the PVP-ethanol solution, and the mixture was stirred at room temperature overnight. The next day, the mixture was condensed and refluxed at 100°C for 6-8h to obtain a black solution, and then the excess solvent was removed by a rotary evaporator to obtain a black product. The black solid was diluted with 15-25mL of pure water and frozen in a -80°C refrigerator. It was then dried in a vacuum freeze dryer for 3-5 days to obtain a black powder, which was stored at room temperature for later use. Using metallic iridium as the raw material, Ir NPs-PVP was prepared by an improved ethanol reduction strategy. After a series of reactions, the final black powder obtained was Ir NPs-PVP ( Figure 1 a). Temperature transmission electron microscopy (TEM) images show that infrared IrNPs-PVP is a subspherical structure ( Figure 1 b).

[0065] 3. Preparation of ETDC-EM / Ir NPs-PVP

[0066] On the 8th day of ETDC culture, ETDCs were collected from the six-well plates and cultured at 2×10 6 Each cell was taken as one unit. ETDC was resuspended in 1 mL of Ir NPs-PVP (0.2 mg / ml, 0.5 mg / ml, 1 mg / ml and 2 mg / ml), and the resuspended liquid was passed through a 1 μm polycarbonate filter membrane using a liposome extruder. The obtained sample was centrifuged at 20,000 g for 30 min, the supernatant was removed, and the precipitate was collected to obtain exosome mimics loaded with Ir NPs-PVP (ETDC-EM / Ir NPs-PVP), which were stored at -80°C.

[0067] In addition, the solution for resuspending cells was replaced with sterile PBS, and the remaining steps were the same to prepare exosome mimics (ETDC-EM) without Ir NPs-PVP.

[0068] Flow cytometry analysis confirmed that the cells expressed the DC marker CD11c, indicating that DC cells were successfully extracted. TEM images showed that ETDC-EM was a spherical structure of 80 to 100 nm with an intact membrane structure ( Figure 1 c), ETDC-EM / Ir NPs-PVP contains Ir NPs-PVP nanoparticles based on the original spherical structure ( Figure 1 d). Statistical analysis of 600 Ir NPs-PVP particles on the TEM image showed an average particle size of 1.5 nm ( Figure 1 e), and the particle size of ETDC-EM measured by a nanoparticle size analyzer was 80 ~ 100 nm, while the particle size of ETDC-EM / Ir NPs-PVP was almost the same as that of ETDC-EM, indicating that there was no significant effect on the vesicle size before and after loading Ir NPs-PVP ( Figure 1 f). In addition, the zeta potential of Ir NPs-PVP in aqueous solution is -15 mV, the zeta potential of ETDC-EM is -11 mV, and the zeta potential of ETDC-EM / Ir NPs-PVP is -23 mV. The change in potential indicates that Ir NPs-PVP is successfully combined with ETDC-EM ( Figure 1 g). The UV-visible spectrum of ETDC-EM / Ir NPs-PVP combines the absorption peaks of Ir NPs-PVP and ETDC-EM, which indirectly verifies that ETDC-EM successfully encapsulates Ir NPs-PVP ( Figure 1 h). In addition, X-ray photon electron spectroscopy (XPS) characterized the chemical state and surface properties of Ir NPs-PVP, confirming the successful coordination of the pyrrole part of PVP with Ir nanozymes ( Figure 1 i).

[0069] Example 2 In vitro antioxidant capacity and ROS scavenging capacity of Ir NPs-PVP:

[0070] Electron spin resonance (ESR) experiments were used to study the scavenging ability of Ir NPs-PVP on ·OH free radicals. 2 O 2 After being irradiated by ultraviolet light, ·OH free radicals are generated. The generated spin adduct DMPO / ·OH has ESR activity, and catalase decomposes H 2 O 2 Can reduce UV-induced H2 O 2 Therefore, the characteristic peak of ·OH radicals on the ESR spectrum disappears, indicating that Ir NPs-PVP can effectively inhibit ·OH radicals ( Figure 2 a).

[0071] The catalase-like (CAT-like) enzyme activity of Ir NPs-PVP was detected by a peroxidase assay kit. 2 O 2 Under the same concentration, with the increase of Ir NPs-PVP concentration, H 2 O 2 The more that is cleared ( Figure 2 b); At the same Ir NPs-PVP concentration (200 μg / ml), as H 2 O 2 With the increase of concentration, Ir NPs-PVP scavenged H 2 O 2 The ability to increase indicates that with the increase of H 2 O 2 With the increase of concentration, the CAT-like activity of Ir NPs-PVP can remove H 2 O 2 Helpful ( Figure 2 c), demonstrating that Ir NPs-PVP has good CAT-like activity.

[0072] At the same time, the catalase activity of Ir NPs-PVP was detected using a catalase activity detection kit, and it can be seen that the catalase-like activity of Ir NPs-PVP is positively correlated with its concentration. The POD activity of Ir NPs-PVP was detected using a peroxidase (POD) detection kit, and it can be seen that the POD-like enzyme activity of Ir NPs-PVP increases with the increase of Ir NPs-PVP concentration ( Figure 2 e).

[0073] Evaluation of the ability of Ir NPs-PVP to scavenge reactive oxygen species. Different concentrations of Ir NPs-PVP were used as experimental groups and 5 mM H 2 O 2 , DCFH-DA, which is a marker of intracellular ROS, stimulated RAW264.7 cells, and flow cytometry was used to detect the fluorescence intensity of intracellular ROS in each group. The results showed that the ROS level was significantly increased after H2O2 stimulation of RAW264.7 cells without Ir NPs-PVP pretreatment. After Ir NPs-PVP pretreatment of RAW264.7 cells, the ROS level was significantly decreased, and there was no statistically significant difference compared with the control group ( Figure 2 f).

[0074] Taken together, these results indicate that Ir NPs-PVP has multi-enzyme mimetic functions, scavenges ROS, and exerts cytoprotective effects.

[0075] Example 3 Evaluation of the anti-inflammatory effect of ETDC-EM:

[0076] The RAW264.7 inflammation model induced by high-dose lipopolysaccharide (LPS) was used to study the anti-inflammatory effect of ETDC-EM. After 1μg / ml LPS acted on RAW264.7 cells for 24h, different concentrations of ETDC-EM were co-incubated with the cells overnight. RAW264.7 cells without ETDC-EM were used as the positive control group, and RAW264.7 cells without LPS and ETDC-EM were used as the blank control group. After collecting the supernatant of each group of cells, enzyme-linked immunosorbent assay (ELISA) was used to study the anti-inflammatory effect of ETDC-EM, and the fluorescence intensity of CD 86 in each group of cells was further detected by flow cytometry.

[0077] The anti-inflammatory effect of ETDC-EM was detected by enzyme-linked immunosorbent assay (ELISA). Figure 3 a. Figure 3 b), flow cytometry was used to detect the fluorescence intensity of CD86 in each group of cells ( Figure 3 c). It can be seen that the levels of intracellular TNF-α and IL-1β in the positive experimental group without ETDC-EM increased significantly, and the expression rate of CD86 positive (a typical marker of M1 macrophages) increased significantly, while ETDC-EM was able to reduce the levels of intracellular TNF-α and IL-1β and the expression rate of CD86 positive. The above results show that ETDC-EM has good anti-inflammatory ability and has a certain protective effect on cells.

[0078] Example 4 Loading and release study of Ir NPs-PVP and biodistribution of ETDC-EM and Ir NPs-PVP in mice:

[0079] 0.2 mg / ml, 0.5 mg / ml, 1 mg / ml and 2 mg / ml of Ir NPs-PVP were mixed with ETDC (2×10 6 After mixing, the mixture was passed through a polycarbonate membrane through a liposome extruder. The resulting solution was centrifuged and the supernatant was removed. The precipitate was measured by ICP-MS for the content of Ir NPs-PVP loaded into ETDC-EM ( Figure 4a), the results show that the loading rate is proportional to the concentration of Ir NPs-PVP stock solution. When the concentration of Ir NPs-PVP is 2 mg / ml, the Ir NPs-PVP loaded into ETDC-EM is 179.263 μg / ml, which is close to 200 μg / ml. Finally, 2 mg / ml Ir NPs-PVP solution and ETDC (2×10 6 The ETDC-EM / Ir NPs-PVP obtained by extruding the ETDC-EM / Ir NPs-PVP together with 10 cells was used as a unit treatment dose in the entire experiment (if not otherwise specified, all the following experiments have this treatment dose).

[0080] The prepared ETDC-EM / Ir NPs-PVP was centrifuged and the supernatant was removed. PBS was slowly added and the mixture was incubated at 37°C. The supernatant was taken at 0h, 1h, 2h, 3h, 4h, 5h, and 6h, and the release of Ir NPs-PVP in vitro was measured by UV spectrophotometer ( Figure 4 b) The results showed that the release amount of Ir NPs-PVP in ETDC-EM / Ir NPs-PVP was proportional to time. The release was relatively slow in 0-5h, and the release increased sharply at 6h.

[0081] One unit of ETDC-EM / Ir NPs-PVP was injected into ALF mice. The mice were killed at 0.5h, 1h, 2h, 3h, 4h, 5h, and 6h, and their liver tissues were collected. The metabolism of Ir NPs-PVP in the liver of mice was determined by ICP-MS. The results showed that Ir NPs-PVP was gradually metabolized in the liver over time, and the metabolism of IrNPs-PVP encapsulated with ETDC-EM was slightly slower ( Figure 4 c).

[0082] The metabolism of ETDC-EM in ETDC-EM / Ir NPs-PVP in ALF mice was analyzed. After injection of ETDC-EM or ETDC-EM / Ir NPs-PVP via the tail vein, in vivo imaging of ALF mice was performed at 1h, 2h, 3h, 4h, 5h, and 6h ( Figure 4 d, e), it is not difficult to find that ETDC-EM or ETDC-EM / Ir NPs-PVP began to accumulate in the liver 1 hour after intravenous injection, reached the highest level at 5 hours, and then began to decline. However, compared with ETDC-EM, ETDC-EM / Ir NPs-PVP accumulated faster in the liver, which is more conducive to timely prevention and treatment of ALF.

[0083] Example 5 Biocompatibility evaluation of TDC-EM / Ir NPs-PVP:

[0084] The hemolysis rate of ETDC-EM / Ir NPs-PVP in mice was detected. The experimental results showed that the red blood cells in the negative control group and the experimental group did not undergo obvious hemolysis, while the red blood cells in the positive control group were completely ruptured, as shown in the photo ( Figure 5 a), the hemolysis rate of each experimental group of ETDC-EM / Ir NPs-PVP was still far below 5% ( Figure 5 b).

[0085] In order to evaluate the biosafety of ETDC-EM / Ir NPs-PVP, the cell viability after ETDC-EM / IrNPs-PVP culture was detected using CCK-8 kit ( Figure 5 c). 0.2 mg / ml, 0.5 mg / ml, 1 mg / ml and 2 mg / ml of Ir NPs-PVP were selected to react with ETDC (2×10 6 After mixing with ETDC-EM / Ir NPs-PVP, the mixture was passed through a polycarbonate membrane through a liposome extruder to obtain 4 different concentrations of ETDC-EM / Ir NPs-PVP. The above-obtained ETDC-EM / Ir NPs-PVP was mixed with RAW264.7 cells for 72 hours, and the cell survival rate was detected every 24 hours. It can be seen that the survival rate of RAW264.7 cells after mixed culture with ETDC-EM / Ir NPs-PVP for 72 hours was close to 100%, which indicates that ETDC-EM / Ir NPs-PVP has good cell biocompatibility.

[0086] The prepared ETDC-EM / Ir NPs-PVP were mixed with RAW264.7 cells. PVP was mixed with RAW264.7 cells for 72 h, and the cell survival rate was detected every 24 h. It can be seen that the survival rate of RAW264.7 cells was close to 100% after ETDC-EM / Ir NPs-PVP was mixed for 72 h, indicating that ETDC-EM / Ir NPs-PVP has good cell biocompatibility.

[0087] Finally, the toxicity of ETDC-EM / Ir NPs-PVP in mice was tested by injecting 100 μL of one unit of ETDC-EM / Ir NPs-PVP. Ir NPs-PVP, ETDC-EM or ETDC-EM / Ir NPs-PVP were injected into healthy mice via the tail vein, and the control group mice were injected with an equal amount of PBS solution. The main organ samples were collected 6 hours after injection, and the H&E staining images of the main organs ( Figure 5d) showed that there were no toxic side effects in the heart, liver, spleen, lung, and kidney of mice treated with Ir NPs-PVP, ETDC-EM, or ETDC-EM / Ir NPs-PVP. All these results ensured the feasibility of subsequent treatment via tail vein injection of ETDC-EM / Ir NPs-PVP.

[0088] Example 6 The therapeutic effect of ETDC-EM / Ir NPs-PVP on acute liver failure (ALF):

[0089] The mouse acute liver failure (ALF) model was established using the classic LPS / D-GalN induction method. C57BL / 6 male mice weighing 20g-25g were selected. Each mouse required 600mg / kg of D-galactosamine and 10mg / kg of LPS, dissolved in normal saline for intraperitoneal injection. After modeling, it was observed that the mice slowed down their activities, ate less, and their feces became thinner. 6 hours after the mouse modeling, the mouse liver was removed, and it can be observed that the congestion of the mouse liver tissue was significantly reduced after treatment, while the liver of the untreated ALF mouse was obviously congested and swollen ( Figure 6 a).

[0090] 40 male C57BL / 6 mice were randomly divided into 4 groups, 3 groups were intraperitoneally injected with LPS / D-GalN, and the last group was intraperitoneally injected with an equal volume of PBS. To verify the protective effect of ETDC-EM / Ir NPs-PVP on ALF mice, 100 μl of Ir NPs-PVP, ETDC-EM or ETDC-EM / Ir NPs-PVP were injected through the tail vein in the three experimental groups, and the control group was injected with an equal volume of PBS. The survival time of the mice was observed and recorded. All mice in the control group died within 12 h, 6 mice injected with IrNP-PVP died, 5 mice injected with ETDC-EM died, and 3 mice injected with ETDC-EM / IrNP-PVP died, which further confirmed the protective effect of ETDC-EM / Ir NPs-PVP on ALF mice. As can be seen from the liver volume, the liver appearance of mice treated with ETDC-EM / IrNP-PVP is closest to that of healthy mice ( Figure 6 a).

[0091] The therapeutic effect of ETDC-EM / Ir NP-PVP was studied by HE staining of liver tissue. Figure 6b). Thirty male C57BL / 6 mice were randomly divided into five groups: a healthy mouse blank control group, an ALF group (injected PBS via tail vein), an experimental group (1) (injected Ir NPs-PVP via tail vein), an experimental group (2) (injected ETDC-EM via tail vein), and an experimental group (3) (injected ETDC-EM / Ir NPs-PVP via tail vein). After the ALF model was successfully established, PBS, Ir NPs-PVP, ETDC-EM, and ETDC-EM / Ir NPs-PVP were immediately injected through the tail vein. Compared with the healthy mouse blank control group, the liver of the ALF group showed extensive tissue damage and a large number of inflammatory cell infiltrations, while the liver damage and inflammatory cell infiltrations of the other three experimental groups were significantly reduced, especially in the ETDC-EM / Ir NPs-PVP group.

[0092] Furthermore, we detected the levels of alanine aminotransferase (AST) and alanine aminotransferase (ALT) in serum to investigate the therapeutic effect of ETDC-EM / Ir NPs-PVP. Blood biochemical analysis showed that the levels of AST and ALT in ALF mice treated with ETDC-EM / Ir NPs-PVP were significantly decreased compared with those in other groups of mice ( Figure 6 c, Figure 6 d). All the results indicated that ETDC-EM / Ir NPs-PVP had the potential to mitigate ALF.

[0093] To further investigate the antioxidant effect of Ir NPs-PVP in vivo, ALF mice were used as a model to test its antioxidant effect. Mouse liver tissue was stained with dihydroethylenediamine (DHE) to assess the level of ROS in the tissue ( Figure 7 ). After treatment with ETDC-EM / Ir NPs-PVP, the red fluorescence in the liver tissue of mice was significantly weakened, indicating that the ROS level in the liver tissue of ALF mice was significantly inhibited after treatment. The above results prove that Ir NPs-PVP still has good antioxidant capacity after being loaded with ETDC-EM in vivo. In order to further prove the anti-inflammatory ability of ETDC-EM / Ir NPs-PVP, TNF-α and IL-1β immunohistochemical staining of liver tissues of mice in each group showed that compared with the ALF control group, the levels of inflammatory factors TNF-α and IL-1β in the three experimental groups were reduced ( Figure 7 a, Figure 7 b) Similarly, the ETDC-EM / IrNP-PVP group had the lowest level of inflammatory factors. Immunohistochemical staining intensity quantification can also directly show that ETDC-EM / Ir NPs-PVP reduced the levels of TNF-α and IL-1β in liver tissue ( Figure 7 c, Figure 7d), indicating that ETDC-EM / Ir NPs-PVP can reduce inflammation in ALF mice in vivo. In addition, Elisa kits were used to detect the expression levels of IL-1β and TNF-α in the serum of each group ( Figure 7 e, Figure 7 f), the expression levels of IL-1β and TNF-α in the serum of the ETDC-EM / Ir NPs-PVP group were also significantly lower than those of the other groups. Next, we further studied the antioxidant capacity of ETDC-EM / Ir NPs-PVP in ALF mice. Dihydroethidium (DHE) staining of mouse liver tissue was used to evaluate the level of ROS in the tissue ( Figure 7 g). After ETDC-EM / Ir NPs-PVP treatment, the red fluorescence was significantly weakened, indicating that the ROS level in the liver tissue of ALF mice was significantly inhibited after treatment. The above results show that Ir NPs-PVP still has good antioxidant capacity in vivo after loading ETDC-EM.

[0094] The above disclosure is only the preferred embodiment of the present invention, which certainly cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.

Claims

1. A biomimetic exosome derived from endotoxin-tolerant dendritic cells loaded with iridium nanozymes, characterized in that: The method comprises bionic exosomes derived from endotoxin-tolerant dendritic cells and iridium nanozyme particles, wherein the iridium nanozyme particles are embedded in the bionic exosomes; The preparation method comprises the following steps: (1) Preparation of endotoxin-tolerant dendritic cells; (2) Preparation of iridium nanozyme particles by ethanol thermal reduction method; (3) resuspending the endotoxin-tolerant dendritic cells prepared in step (1) with the dispersion prepared with the iridium nanozyme particles prepared in step (2), passing the resuspended liquid through a 1 μm polycarbonate filter membrane using a liposome extruder, centrifuging the extruded sample at 20,000 g for 30 min, removing the supernatant, and collecting the precipitate to obtain bionic exosomes derived from endotoxin-tolerant dendritic cells loaded with iridium nanozyme; The dendritic cells are dendritic cells derived from mouse bone marrow; Step (3) includes the following steps: 2×10 6 Each cell was taken as one unit and resuspended in 1 mL of dispersion solution. The amount of iridium nanozyme particles in the dispersion solution was 2 mg / mL.

2. The endotoxin-tolerant dendritic cell-derived biomimetic exosomes loaded with iridium nanozymes according to claim 1, characterized in that: Step (1) includes the following steps: Dendritic cells were added to RPMI-1640 complete medium containing GM-CSF and IL-4 cytokines, seeded in well plates, and cultured in a 37°C constant temperature incubator; On the 4th day of culture, the culture supernatant in the well plate was aspirated, and RPMI-1640 complete medium containing GM-CSF and IL-4 cytokines was added again; On the 5th day of culture, 100 ng / mL LPS was added for stimulation for 48 h. On the 7th day of culture, the culture supernatant in the well plate was aspirated, and fresh RPMI-1640 complete medium containing GM-CSF and IL-4 cytokines was added, and 1 μg / mL LPS was added for stimulation for 24 h to obtain endotoxin-tolerant dendritic cells.

3. The endotoxin-tolerant dendritic cell-derived biomimetic exosomes loaded with iridium nanozymes according to claim 1, characterized in that: Step (2) includes the following steps: IrCl3·3H2O was dissolved in pure water and sonicated in an ultrasonicator for more than 3 h to obtain a transparent light yellow solution; Then, PVP was dissolved in the ethanol solution and mixed well. Finally, under magnetic stirring, the IrCl3·3H2O aqueous solution was slowly dropped into the PVP-ethanol solution, and the mixture was stirred at room temperature overnight; The next day, the mixture was condensed and refluxed at 100° C. for 6-8 h to obtain a black solution, and then excess solvent was removed by a rotary evaporator to obtain a black product, which was then dried using a vacuum freeze dryer to obtain iridium nanozyme particles.

4. Use of the bionic exosomes derived from endotoxin-tolerant dendritic cells loaded with iridium nanozymes as claimed in claim 1 to prepare a drug for treating acute liver failure.

5. A drug for treating acute liver failure, characterized in that: It comprises the bionic exosomes derived from endotoxin-tolerant dendritic cells loaded with iridium nanozyme as claimed in claim 1.

6. The drug for treating acute liver failure according to claim 5, characterized in that: The medicine is an injection preparation.

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