Preparation method and application of engineered exosome for targeted regulation of liver macrophages

Abstract

The invention discloses a preparation method and application of an engineered exosome for targeted regulation and control of liver macrophages. The human umbilical cord-derived mesenchymal stem cell exosome capable of loading vitamin D3 is constructed, and the phagocytosis of macrophages is utilized to activate AhR receptors of the macrophages, so that the phenotypes and functions of the macrophages are regulated and controlled. The engineered exosome can reprogram M1 type macrophages into M2 type macrophages, reduce inflammatory cytokines of TNF-alpha, INF-gamma, IL-1beta and IL-6, and can promote release of IL-22. IL-22 can improve apoptosis of liver cells and promote proliferation of the liver cells. In-vivo experiments show that the engineered exosome can obviously improve the autoimmune hepatitis symptom, has no damage to organs of the whole body, can overcome side effects brought by traditional glucocorticoid and immunosuppressive drugs, and has wide application prospects and clinical values.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a method for preparing engineered exosomes for targeted regulation of liver macrophages and applications thereof. Background Art

[0002] Autoimmune hepatitis (AIH) is a chronic inflammatory liver disease caused by autoimmune system disorders, which can rapidly progress to cirrhosis and liver failure. AIH is characterized by autoimmune cells attacking hepatocytes, and is characterized by positive serum autoantibodies and elevated immunoglobulin G levels. The main features of liver histology are interface hepatitis and rosette-like changes in hepatocytes. At the same time, 68% to 81% of AIH patients have insufficient vitamin D3 (VD3 < 30nmol / l), and about 20% of AIH patients have severe vitamin D3 deficiency (VD3 < 25nmol / l). Severe vitamin D3 deficiency is closely related to adverse events such as unresponsiveness to AIH treatment and disease progression.

[0003] At present, nonspecific immunosuppressants (prednisone) combined with azathioprine or prednisone are mainly used as the standard treatment for AIH. However, at least 10% to 15% of patients respond poorly to standard treatments, and some patients relapse due to intolerance to drug side effects or discontinuation of medication. Long-term use of glucocorticoids can cause obvious adverse reactions, such as Cushing's syndrome and osteoporosis. As the incidence of AIH increases year by year, some patients show poor response or intolerance to standard treatment, which increases the difficulty of AIH treatment. Therefore, AIH is in urgent need of new treatment strategies and methods.

[0004] Umbilical cord-derived mesenchymal stem cells (UC-MSC) are a type of multipotent stem cell with the characteristics of low immunogenicity, easy access, easy in vitro culture, and multidirectional differentiation potential. They have been clinically used to treat a variety of immune-related diseases such as graft-versus-host disease and refractory systemic lupus erythematosus. In recent years, more and more evidence has shown that UC-MSC has a positive effect in the treatment of liver diseases. Its anti-fibrosis, immunomodulatory, and hepatocyte differentiation properties make it a new method for treating liver diseases. UC-MSC mainly exerts its immunoregulatory function by directly interacting with different types of immune cells and releasing various immunoregulatory active molecules or exosomes containing different active factors.

[0005] Exosomes are nano-sized vesicles with a lipid bilayer membrane structure and a diameter of 30-200nm, which can carry important information such as various proteins, lipids and RNA of the mother cell. Exosomes derived from UC-MSC can regulate innate and adaptive immune responses, inhibit T lymphocyte function, reduce B cell activation, proliferation and secretion, affect macrophage differentiation and dendritic cell maturation, and inhibit the cytotoxic activity of natural killer cells to exert their immunoregulatory effects, thereby improving the lifespan of UC-MSC exosome-derived drug delivery carriers and the bioavailability of drugs. Moreover, more and more evidence shows that UC-MSC can produce a large number of exosomes compared to other cells. Therefore, exosomes derived from UC-MSC have excellent properties.

[0006] VD3 is a fat-soluble vitamin. It is converted in vivo through the thermal isomerization of 7-dehydrocholesterol on the surface of the skin under ultraviolet radiation. It binds to VD3 binding protein (DPD) and is transported to the liver. Under the action of 25-hydroxylase (CYP2R1), 25(OH)VD3 is generated, which is further transported to the kidneys and 1α-hydroxylase (CYP27B1) is used in the proximal tubules to produce its final active form, calcitriol (1,25(OH)2VD3). More and more studies have shown that VD3 not only has classical pathways for regulating bone metabolism and calcium homeostasis, but also non-classical pathways for immune-mediated, anti-oxidant, anti-fibrotic and anti-inflammatory effects.

[0007] Among mammalian tissues and organs, liver-resident macrophages account for the largest proportion and are important immune cells that regulate the liver's natural immune advantage. They play an important role in maintaining liver immune tolerance homeostasis and imbalance. Therefore, targeting macrophages to treat autoimmune hepatitis is an ideal new strategy.

[0008] By constructing engineered exosomes that can target macrophages, regulating the phenotype and function of macrophages, and then improving damaged liver cells for the treatment of AIH, this provides a new strategy for the treatment of AIH. Summary of the invention

[0009] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of the above-mentioned problems and provide a method for preparing engineered exosomes for targeted regulation of liver macrophages and its application.

[0010] The first object of the present invention is to provide a method for preparing engineered VD3-loaded exosomes.

[0011] The second object of the present invention is to provide an engineered exosome capable of targeting and regulating macrophages.

[0012] The third object of the present invention is to provide the use of the engineered exosomes in AIH.

[0013] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0014] The present invention provides a method for preparing engineered exosomes for targeted regulation of liver macrophages and its application, which specifically comprises the following steps:

[0015] S1: The UC-MSC culture supernatant was extracted by ultracentrifugation, and the precipitate was exosomes.

[0016] S2: The exosomes obtained in step S1 are loaded with VD3 by co-incubation, and free VD3 is removed by ultracentrifugation.

[0017] S3: The engineered exosomes obtained in step S2 are applied to the targeted regulation of macrophages and the treatment of autoimmune diseases.

[0018] Preferably, the exosomes in step S1 are derived from the culture supernatant of P4 to P6 stem cells and are obtained by ultracentrifugation.

[0019] Preferably, the engineered exosomes in step S2 are obtained by co-incubating exosomes with VD3.

[0020] Preferably, the co-incubation conditions in step S2 are: 200 μg of exosomes and 100 μg of VD3 are incubated in an oscillator (37° C., 190 rpm, 2 h). Finally, free VD3 is removed by ultracentrifugation.

[0021] The present invention provides an engineered VD3-loaded exosome, which is prepared by the above method.

[0022] The present invention provides the use of the engineered exosomes for targeted regulation of liver macrophages in drugs for treating autoimmune hepatitis.

[0023] The present invention has the following beneficial effects:

[0024] The present invention provides an engineered exosome that targets and regulates liver macrophages, utilizes the phagocytosis of macrophages, activates the AhR receptor of macrophages, and then regulates the phenotype and function of macrophages. The engineered exosomes can reprogram M1 macrophages into M2 macrophages, reduce TNF-α, INF-γ, IL-1β and IL-6 inflammatory cytokines, and promote the release of IL-22. IL-22 can improve the apoptosis of liver cells and promote their proliferation. In vivo experiments have shown that engineered exosomes can significantly improve the symptoms of autoimmune hepatitis, have no damage to systemic organs, can overcome the side effects of traditional glucocorticoids and immunosuppressive drugs, and have broad application prospects and clinical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1This is a transmission electron microscopy image of UC-MSC-derived exosomes extracted in Example 1;

[0026] Figure 2 This is a diagram of the particle size of the engineered exosomes in Example 1;

[0027] Figure 3 This is the analysis diagram of the drug loading of engineered exosomes determined by HPLC in Example 2;

[0028] Figure 4 Confocal images of engineered exosomes taken up by macrophages;

[0029] Figure 5 The engineered exosomes stimulate AhR receptors of macrophages and promote IL22 expression;

[0030] Figure 6 To engineer exosomes to regulate the polarization state of macrophages;

[0031] Figure 7 Engineered exosomes to reduce macrophage inflammatory cytokines;

[0032] Figure 8 This is a flow cytometric analysis of apoptotic hepatocytes using the supernatant of macrophages treated with engineered exosomes;

[0033] Fig. 9 Immunofluorescence image showing that the supernatant of macrophages treated with engineered exosomes promoted hepatocyte proliferation;

[0034] Fig.10 is the in vivo tissue distribution map of engineered exosomes;

[0035] Fig.11 The figure shows that engineered exosomes reduce the ALT and AST levels in AIH serum;

[0036] Fig.12 Analysis of liver HE pathology in AIH treated with engineered exosomes;

[0037] Fig.13 TUNEL images of liver for improving AIH by engineered exosomes;

[0038] Fig.14 HE pathological analysis for tissue safety evaluation of engineered exosomes; DETAILED DESCRIPTION

[0039] The principles and features of the present invention are described below in conjunction with examples, which are only used to explain the present invention and are not used to limit the scope of the present invention. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0040] Example 1. Extraction and identification of exosomes from UC-MSCs

[0041] 1. Extraction of exosomes from UC-MSC

[0042] The culture supernatant of P4-P5 UC-MSCs was collected and centrifuged at 330 g for 10 min to remove dead cells; at 2,000 g for 10 min to remove cell debris; at 10,000 g for 30 min to further remove cell debris and large particles; the supernatant was filtered through a 0.22 μm microporous filter to remove vesicles with larger particle sizes; ultra-high-speed centrifugation was performed at 10,0000 g for 2 h, the centrifuge tube was gently removed, the culture medium was carefully aspirated, and the white precipitate at the bottom of the tube was the exosomes. 1 mL of sterile saline was added for reconstitution and placed in a -80 refrigerator for later use.

[0043] 2. Identification of exosomes

[0044] The exosomes prepared in this example were photographed using a transmission electron microscope. Figure 1 As shown in Figure 2, the exosome morphology is a saucer-shaped vesicle structure. The exosome particle size was characterized by the nanoparticle tracking instrument NanoSight. Figure 2 As shown, the exosome particle size is concentrated around 150 nm, which is consistent with the size range of exosomes.

[0045] Example 2. Engineering modification of exosomes loaded with VD3

[0046] 1. Exosome co-incubation with VD3

[0047] Since VD3 is extremely insoluble in water, VD3 loading was performed by co-incubation. The co-incubation conditions were as follows: 200 μg of exosomes and 100 μg of VD3 were incubated on a shaker (37°C, 190 rpm, 2 h), and finally free VD3 was removed by ultracentrifugation (100,000 g, 1 h).

[0048] 2. Determination of exosome drug loading

[0049] The drug loading capacity of the engineered exosomes prepared in this example was measured. The engineered exosomes were dissolved in methanol, centrifuged to remove the protein, and finally the supernatant was dried and the drug loading capacity of the exosomes was detected by HPLC. Figure 3 As shown, the drug loading of exosomes was 1.05%.

[0050] Example 3. Engineered exosomes target and regulate the phenotype and function of macrophages

[0051] 1. Macrophages engulf engineered exosomes

[0052] The engineered exosomes prepared in Example 2 were subjected to in vitro functional studies. The exosomes were stained with DIO and the phagocytosis of exosomes by RAW264.7 macrophages was observed using a confocal instrument. Figure 4 As shown, RAW264.7 macrophages can phagocytize large amounts of engineered exosomes.

[0053] 2. Engineered exosomes stimulate AhR receptors in macrophages

[0054] The engineered exosomes prepared in Example 2 were subjected to in vitro functional studies. The exosomes were co-cultured with RAW264.7 macrophages, and the AhR receptor downstream genes were detected using q-PCR technology. Figure 5 As shown, engineered exosomes can stimulate the expression of AhR receptor downstream genes CYP1A1, CYP1A2 and IL22 in macrophages, accompanied by a dose-dependent effect.

[0055] 3. Effects of engineered exosomes on macrophage phenotype

[0056] The engineered exosomes prepared in Example 2 were subjected to in vitro functional studies. M1 macrophages were induced by LPS, and the effects of engineered exosomes on macrophage phenotypes were analyzed using q-PCR technology and flow cytometry. Figure 6 and Figure 7 As shown, engineered exosomes can significantly reduce the M1 polarization phenotype and reduce the mRNA levels of inflammatory cytokines TNF-α, IFN-γ, IL-1β, and IL-6.

[0057] 4. Engineered exosomes inhibit hepatocyte apoptosis

[0058] The engineered exosomes prepared in Example 2 were subjected to in vitro functional studies. The engineered exosomes were co-cultured with RAW264.7 macrophages, and after 24 hours, the culture supernatant was co-cultured with AML 12 hepatocytes, and ConA (20 μg / ml) was added to induce an in vitro liver injury model, and the apoptosis of hepatocytes was detected by flow cytometry. Figure 8 As shown, the supernatant of co-culture of macrophages and engineered cells can effectively reduce the apoptosis of hepatocytes.

[0059] 5. Engineered exosomes promote hepatocyte proliferation

[0060] The engineered exosomes prepared in Example 2 were subjected to in vitro functional studies. The engineered exosomes were co-cultured with RAW264.7 macrophages, and after 24 hours, the culture supernatant was co-cultured with AML12 hepatocytes, and ConA (20 μg / ml) was added to induce an in vitro liver injury model. TM EdU-594 cell proliferation detection kit and immunofluorescence microscopy were used for detection. Fig. 9 As shown, the supernatant of co-culture of macrophages and engineered cells can effectively promote hepatocyte proliferation.

[0061] Example 4. Application of engineered exosomes in the treatment of autoimmune hepatitis

[0062] 1. In vivo tissue distribution of engineered exosomes

[0063] The engineered exosomes prepared in Example 2 were subjected to in vivo tissue distribution studies. 100 μg of DIR-labeled engineered exosomes were injected into the tail vein of C57BL / 6J mice, and in vivo imaging was performed 24 hours later. Fig.10 As shown, the engineered exosomes were mainly enriched in liver tissue.

[0064] 2. In vivo efficacy evaluation of engineered exosomes

[0065] The engineered exosomes prepared in Example 2 were subjected to in vivo functional studies. ConA (20 mg / kg) was injected into the tail vein of C57BL / 6J mice, and 100 μg of engineered exosomes was injected into the tail vein 2 hours later. The mice were dissected 24 hours later. The changes of ALT and AST aminotransferases in the mice were detected using a kit. Fig.11 As shown in Figure 2, engineered exosomes can significantly reduce the levels of ALT and AST in AIH animal models. HE staining was used to analyze liver pathology, such as Fig.12 As shown in Figure 2, engineered exosomes can significantly improve the inflammatory cell infiltration and interface hepatitis pathological characteristics of AIH liver. TUNEL staining was used to analyze the apoptosis of the liver. Fig.13 As shown, engineered exosomes can significantly reduce apoptosis in AIH liver.

[0066] 3. Safety evaluation of engineered exosomes

[0067] The engineered exosomes prepared in Example 2 were evaluated for safety. 100 μg of engineered exosomes were injected into the tail vein of normal C57BL / 6J mice, and the mice were dissected 24 hours later, and the liver, spleen, heart, kidney and lung tissues were collected for histopathological analysis using HE staining. Fig.14 As shown, the engineered exosomes had no damaging effect on the above-mentioned tissues, which preliminarily verified the in vivo biosafety of the drug.

[0068] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing engineered exosomes for targeted regulation of liver macrophages and its application, characterized in that: The specific steps include: S1: The culture supernatant of human umbilical cord-derived mesenchymal stem cells (UC-MSC) was extracted by ultracentrifugation, and the precipitate was exosomes. S2: The exosomes obtained in step S1 are loaded with vitamin D3 (VD3) by co-incubation, and free VD3 is removed by ultracentrifugation. S3: The engineered exosomes obtained in step S2 are applied to the targeted regulation of macrophages and the treatment of autoimmune diseases.

2. The method according to claim 1, characterized in that: Exosomes were obtained from the culture supernatant of P4 to P6 UC-MSCs by ultracentrifugation.

3. The method according to claim 1, characterized in that: The engineered exosomes were obtained by co-incubating exosomes with VD3.

4. The method according to claim 1, characterized in that: VD3 is 25(OH)VD3 or (1,25(OH)2VD3) or its derivatives.

5. The method according to claim 1, characterized in that: The co-incubation conditions were as follows: 200 μg of exosomes and 100 μg of VD3 were incubated on a shaker (37° C., 190 rpm, 2 h).

6. The method according to claim 1, characterized in that: The drug loading was 1.05%.

7. A method for preparing engineered exosomes for targeted regulation of liver macrophages and its application, characterized in that: The invention is prepared by the method according to any one of claims 1 to 5.

8. Use of the engineered exosomes according to claim 7 as highly efficient macrophage targeting agents.

9. The engineered exosomes according to claim 7 can stimulate AhR receptors in macrophages and promote the release of IL-22 functional cytokines.

10. Use of the engineered exosomes according to claim 7 in the preparation of a drug for autoimmune hepatitis.

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