Preparation method, preparation and application of mitochondria-derived vesicles

Abstract

The embodiment of the invention provides a preparation method of mitochondrial derived vesicles as well as a preparation and application of the mitochondrial derived vesicles. The preparation method comprises the following steps: feeding SIRT3 plasmids and an LNPs lipid nanoparticle solution into aged Raw264.7 macrophages, extracting mitochondria, incubating, and then extracting the mitochondria derived vesicles. According to the preparation method, the mitochondrial derived vesicles with high-efficiency delivery advantage and treatment effect can be obtained, and the vesicles can regulate and restore macrophage metabolism, so that senescent macrophages are updated, and meanwhile, pro-apoptotic proteins overexpressed in a damaged environment are consumed, and myofibroblast apoptosis is promoted, so that the treatment effect is achieved. The preparation method is simple and can be applied to large-scale clinical application, and the mitochondrial derived vesicles have biological safety, can be used for continuous observation and treatment and have wide clinical application advantages.

Description

Technical Field

[0001] The present invention relates to the field of molecular technology drugs, and more particularly to a preparation method of mitochondrial-derived vesicles, its preparations and applications. Background Art

[0002] Pulmonary fibrosis can lead to macrophage metabolic disorders. Chronic inflammation and oxidative stress are the main driving factors. Inflammatory factors (such as TNF-α, IL-1β) and reactive oxygen species (ROS) damage mitochondrial function, inhibit oxidative phosphorylation, and prompt macrophage metabolism to shift to glycolysis. The activation of the TGF-β signaling pathway further exacerbates metabolic reprogramming, promotes glycolysis and inhibits mitochondrial energy generation. In addition, hypoxia in the pulmonary fibrosis microenvironment induces the expression of HIF-1α, enhancing the expression of glycolysis-related genes. Changes in the extracellular matrix also affect the metabolic adaptability of macrophages. These metabolic disorders not only weaken the immune function of macrophages, but also promote the formation of pro-fibrotic phenotypes and exacerbate the process of pulmonary fibrosis.

[0003] Studies have shown that during the process of pulmonary fibrosis, the expression of SIRT3 (deacetylase 3) is significantly reduced, resulting in a decline in antioxidant stress capacity and impaired mitochondrial function, which in turn exacerbates the fibrotic progression of lung tissue. The reduction of SIRT3 is closely related to oxidative stress, mitochondrial dysfunction and macrophage senescence. To alleviate this situation, restoring the expression of SIRT3 has become a potential therapeutic strategy. However, due to the unique physiological characteristics of macrophages, gene delivery and effective expression face difficulties such as transfection. Summary of the Invention

[0004] In view of this, therefore, in this study, a nanoformulation was first designed for effective delivery of the SIRT3 gene, and a preparation method of mitochondrial-derived vesicles, its preparations and pharmaceutical uses were also provided.

[0005] In the first aspect of the present invention, a preparation method of mitochondrial-derived vesicles is provided. The preparation method of the mitochondrial-derived vesicles includes the following steps:

[0006] a. LNPs lipid nanoparticles are added to a triphenylphosphine solution. After mixing the SIRT3 plasmid and the lipid nanoparticle solution in a ratio of 1:20 to 1:40 for 1-2 h, it is added to senescent macrophages and cultured for another 24 h-48 h; the triphenylphosphine solution is a mixed solution of triphenylphosphine, EDC and NHS;

[0007] b. Then mitochondria are extracted from the macrophages, and the mitochondria and the supernatant in the last step of mitochondrial extraction are incubated at 37 °C for 2-4 hours, and finally mitochondrial-derived vesicles are obtained by ultracentrifugation.

[0008] Further, the triphenylphosphine solution is prepared by stirring triphenylphosphine, EDC, and NHS in a mass ratio of 4:5:6 in a PB solution for 4 hours to activate triphenylphosphine;

[0009] Further, the preparation method of LNPs lipid nanoparticles includes: dissolving co-lipid (D), amphiphilic lecithin (L), cholesterol, DC cholesterol, and liposomal phospholipid CL in an organic solvent, and preparing nanoparticles by the ethanol injection method.

[0010] Further, the co-lipid is selected from 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE); the amphiphilic lecithin is selected from at least one of soybean lecithin, egg yolk lecithin, hydrogenated lecithin, deoxycholine, and isolecithin; the liposomal phospholipid CL is DSPE-xPEGy, where x is selected from at least one of -CH3, -NH2, -COOH, -MAL, -OCH3; and the molecular weight of y is selected from at least one of 1000, 2000, and 5000.

[0011] Further, by mass ratio, the mass ratio of co-lipid: amphiphilic lecithin: cholesterol: DC cholesterol: liposomal phospholipid CL is 2:8:2:6:1.

[0012] Further, the organic solvent is selected from at least one of methanol, ethanol, chloroform, and dichloromethane.

[0013] The base sequence of the SIRT3 gene is shown in SEQ ID No.1.

[0014] Further, the macrophage is Raw 264.7, and other macrophages can also be selected.

[0015] In the second aspect of the present invention, mitochondrial-derived vesicles obtained by any of the preparation methods of the first aspect of the present invention are provided.

[0016] A preparation combining a mitochondrial-derived vesicle prepared by any of the preparation methods of the first aspect of the present invention with the prior art is also provided.

[0017] In the third aspect of the present invention, a pharmaceutical use is provided, including the application of the mitochondrial-derived vesicles described in the second aspect or the preparation containing the mitochondrial-derived vesicles in the preparation of drugs for diseases related to abnormal expression of apoptosis genes.

[0018] Further, the diseases include pulmonary fibrosis, tumors, inflammatory diseases, or metabolic diseases.

[0019] Beneficial effects: The present invention provides a mitochondrial-derived vesicle with high-efficiency delivery advantages and therapeutic effects, having the following advantages:

[0020] 1. The mitochondrial-derived vesicles have good morphology and stable particle size, enabling the vesicles to effectively deliver SIRT3 into cells to achieve the therapeutic effect.

[0021] 2. The mitochondrial-derived vesicles can precisely target mitochondria, have good mitochondrial targeting effect, improve the accuracy of SIRT3 drug delivery, and enable it to play a role, so as to achieve an effective and precise therapeutic effect.

[0022] 3. The mitochondrial-derived vesicles can regulate and restore macrophage metabolism, thereby rejuvenating senescent macrophages, while consuming the overexpressed pro-apoptotic proteins in the damaged environment and promoting the apoptosis of myofibroblasts to achieve the therapeutic effect.

[0023] 4. The preparation process of the mitochondrial-derived vesicles is simple, can be used for large-scale clinical application, and the mitochondrial-derived vesicles have biological safety, can be used for continuous observation and treatment, and have broad clinical application advantages.

[0024] It should be understood that the content described in the Summary of the Invention section is not intended to limit the key or important features of the embodiments of the present invention, nor is it used to limit the scope of the present invention. Other features of the present invention will become easily understood through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Combined with the drawings and referring to the following detailed description, the descriptions, other features, advantages and aspects of the embodiments of the present invention will become more obvious. In the drawings, the same or similar reference numerals represent the same or similar elements, where:

[0026] Figure 1 is the evaluation of the particle size of the LNPs lipid nanoparticles DLNPs@T@SIRT3 according to the embodiments of the present invention.

[0027] Figure 2 is the evaluation of the transfection of the LNPs lipid nanoparticles DLNPs@T@SIRT3 according to the embodiments of the present invention.

[0028] Figure 3 is the evaluation of the uptake of the LNPs lipid nanoparticles DLNPs@T@SIRT3 in cells according to the embodiments of the present invention.

[0029] Figure 4 is the evaluation of the mitochondrial targeting ability of the LNPs lipid nanoparticles DLNPs@T@SIRT3 according to the embodiments of the present invention.

[0030] Figure 5 is the evaluation of the lysosomal escape ability of the LNPs lipid nanoparticles DLNPs@T@SIRT3 according to the embodiments of the present invention.

[0031] Figure 6 is the evaluation of the morphology and particle size of mitochondrial-derived vesicles (MDVs) according to the embodiments of the present invention. (SIRT3+)

[0032] Figure 7 is the evaluation of the targeting effect of mitochondrial-derived vesicles (MDVs) according to the embodiments of the present invention. (SIRT3+)

[0033] Figure 8 is the evaluation of the regulation of macrophages by mitochondrial-derived vesicles (MDVs) according to the embodiments of the present invention. (SIRT3+)

[0034] Figure 9 is the evaluation of the regulation of myofibroblasts by mitochondrial-derived vesicles (MDVs) according to the embodiments of the present invention. (SIRT3+)

[0035] Figure 10 is the evaluation of the accumulation of mitochondrial-derived vesicles (MDVs) at the fibrotic site according to the embodiments of the present invention. (SIRT3+)

[0036] Figure 11 is the evaluation of the therapeutic effect on mice with pulmonary fibrosis by mitochondrial-derived vesicles (MDVs) according to the embodiments of the present invention. (SIRT3+)

[0037] Figure 12 is a schematic diagram of the SIRT3 plasmid. Detailed implementation manners

[0038] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention. For the experimental methods without specific conditions noted in the embodiments, they are generally carried out under conventional conditions or according to the conditions recommended by the manufacturers.

[0039] In addition, the term "and / or" in this article is only a description of the association relationship of associated objects, indicating that there can be three relationships. For example, A and / or B can represent: A exists alone, A and B exist simultaneously, and B exists alone. In addition, the character " / " in this article generally represents an "or" relationship between the associated objects before and after.

[0040] Example 1 Evaluation of the particle size of LNPs lipid nanoparticles DLNPs@T@SIRT3 Preparation method of lipid nanoparticles DLNPs@T@SIRT3:

[0041] 1. Weigh 1 mg of co-lipid DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), 4 mg of lecithin, 1 mg of cholesterol, 3 mg of DC cholesterol, and 0.5 mg of DSPE-PEG 1000 -NH2, and dissolve them separately in 100 - 400 μL of ethanol solution. After complete dissolution, mix all the ethanol solutions and slowly add them to 2 mL of PB solution (phosphate buffer). React overnight, and use an ultrasonic crusher to fully homogenize the nano-preparation for 10 min to form a suspension, then LNPs lipid nanoparticles are obtained;

[0042] 2. Stir triphenylphosphine, EDC (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride), and NHS (N-hydroxysuccinimide) in a PB solution at a mass ratio of 4:5:6 for 4 hours to activate triphenylphosphine; Mix LNPs lipid nanoparticles with the activated triphenylphosphine solution at a mass ratio of DSPE-PEG 1000 -NH2:triphenylphosphine of 5:8 to obtain the nanoparticle solution DLNPs@T;

[0043] 3. Mix the SIRT3 plasmid and the nanoparticle solution at a mass ratio of SIRT3 plasmid:cationic material DC cholesterol in the nanoparticle solution of 1:40 and let it stand at room temperature for 2 h to obtain the lipid nanoparticles DLNPs@T@SIRT3 with SIRT3. The SIRT3 plasmid in this example is from Public Protein / Plasmid Library, catalog number: PPL02422-2b. The schematic diagram of the SIRT3 plasmid is as Figure 12 shown, and other recombinant plasmids containing the SIRT3 gene can also be used. The base sequence of the SIRT3 gene is as follows (SEQ ID No.1):

[0044]

[0045]

[0046] Use a Malvern potentiometric particle size analyzer to measure the particle size of the nanoparticle preparation. The results are as Figure 1 shown, indicating that the particle size of the obtained DLNPs@T@SIRT3 solution is uniform.

[0047] Example 2 Evaluation of the gene transfection of LNPs lipid nanoparticles DLNPs@T

[0048] After obtaining DLNPs@T according to the preparation method described in Example 1, a fluorescent gene plasmid (pDNA) was selected. In this example, GFP was selected for testing. DLNPs@T@pDNA was obtained according to the ratio of the fluorescent gene plasmid to DC cholesterol (1 mg or 2 mg) in DLNPs@T being 1:20 or 1:40 respectively.

[0049] Raw 264.7 macrophages were seeded in a six-well plate at 2×10 5 cells / well and cultured in a cell incubator at 37 °C and 5% CO2 for 24 h. After the Raw 264.7 macrophages completely covered the cell plate, DLNPs@T@pDNA was added. Using lipofectamine3000 as a control, after incubating for 24 h under the culture conditions of 37 °C and 5% CO2, a fluorescence microscope was used to observe the fluorescence intensity in the two groups of cells to evaluate the transfection of different lipid nanoparticles in cells. The results are as Figure 2 shown. The lipid nanoparticles provided by the present invention can have good transfection efficiency in macrophages, and their transfection effect is better than that of the commonly used commercial transfection reagent lipofectamine3000.

[0050] Example 3 Evaluation of the Uptake of LNPs Lipid Nanoparticles DLNPs@T@SIRT3 in Cells

[0051] 1. Weigh 1 mg of co-lipid DOPE, 4 mg of lecithin, 1 mg of cholesterol, 3 mg of DC cholesterol, 0.5 mg of DSPE-PEG1000-NH2, and 0.5 mg of DiI dye and dissolve them separately in 100 - 400 μL of ethanol solution. After complete dissolution, mix all the ethanol solutions and slowly add them to 2 mL of PB solution. React overnight in the dark. After using an ultrasonic disrupter to fully homogenize the nanoformulation for 10 min to form a suspension, lipid nanoparticles are obtained.

[0052] 2. Stir triphenylphosphine, EDC, and NHS in a PB solution at a mass ratio of 4:5:6 for 4 hours to activate triphenylphosphine; mix the lipid nanoparticles with the activated triphenylphosphine solution to obtain the nanoparticle solution DLNPs@T.

[0053] 3. Mix the SIRT3 plasmid and the nanoparticle solution at a mass ratio of SIRT3 plasmid: cationic material DC cholesterol in the nanoparticle solution of 1:40 and let it stand at room temperature for 2 h. Specifically, the mass of the SIRT3 plasmid is 20 μg, and the lipid nanoparticles DLNPs@T@SIRT3 containing DiI dye are obtained.

[0054] Prepare lipid nanoparticles DLNPs@SIRT3 containing DiI dye according to the above steps 1 and 3 without step 2.

[0055] Seed Raw 264.7 macrophages at 5×10 4 cells / well into 2 confocal dishes respectively, and culture them in a cell incubator at 37°C and 5% CO2 for 24 h. After the Raw 264.7 macrophages completely cover the confocal dishes, pour out the culture medium, wash the dishes 3 times with PBS, and then add lipid nanoparticles DLNPs@T@SIRT3 respectively. Continue to incubate them in the cell incubator at 37°C and 5% CO2 for 1 and 4 h respectively. Then, aspirate the culture medium containing the DLNPs@T@SIRT3 solution, wash the culture plates 3 times with PBS, and add 200 μL of Hoechst staining solution to stain the cell nuclei respectively. After staining for 15 min, wash the dishes 3 times with PBS, add 500 μL of culture medium to each dish respectively, and evaluate the uptake of lipid nanoparticles DLNPs@T@SIRT3 in cells at different time points under a confocal microscope.

[0056] The uptake effect of lipid nanoparticles DLNPs@T@SIRT3 measured in this example in Raw 264.7 macrophages is as Figure 3 shown. In Raw 264.7 macrophages, when DLNPs@T@SIRT3 is administered for 4 h, it has a good uptake effect.

[0057] Example 4 Evaluation of the mitochondrial targeting ability of LNPs lipid nanoparticles DLNPs@T@SIRT3

[0058] Seed Raw 264.7 macrophages at 5×10 4 cells / well into 2 confocal dishes respectively, and culture them in a cell incubator at 37°C and 5% CO2 for 24 h. After the Raw 264.7 macrophages completely cover the confocal dishes, pour out the culture medium, wash the dishes 3 times with PBS, and then add lipid nanoparticles DLNPs@T@SIRT3 containing DiI dye prepared as in Example 3 respectively. Continue to incubate them in the cell incubator at 37°C and 5% CO2 for 1 h and 4 h respectively. Then, aspirate the culture medium containing the DLNPs@T@SIRT3 solution, wash the culture plates 3 times with PBS, and add 200 μL of 100 μM mitochondrial green fluorescent probe MitoTracker Green to stain the mitochondria respectively. After staining for 30 min, wash the dishes 3 times with PBS, add 200 μL of Hoechst staining solution to stain the cell nuclei respectively, and the staining time is 15 min; after 15 min, wash the dishes 3 times with PBS, add 500 μL of culture medium to each dish respectively, and evaluate the mitochondrial targeting of lipid nanoparticles DLNPs@T@SIRT3 in cells at different time points under a confocal microscope.

[0059] The mitochondrial targeting effect of the lipid nanoparticles DLNPs@T@SIRT3 measured in this example in Raw 264.7 macrophages is as follows Figure 4 shown. In Raw 264.7 macrophages, when DLNPs@T@SIRT3 is administered for 4 h, it has a good mitochondrial targeting effect.

[0060] Example 5 Evaluation of the lysosomal escape ability of the LNPs lipid nanoparticles DLNPs@T@SIRT3

[0061] Seed Raw 264.7 macrophages at 5×10 4 cells / well into 2 confocal dishes respectively, and culture them in a cell incubator at 37 °C and 5% CO2 for 24 h. After the Raw 264.7 macrophages completely cover the confocal dishes, pour out the culture medium, wash the dishes 3 times with PBS, and then add the lipid nanoparticles DLNPs@T@SIRT3 containing DiI dye prepared in Example 3 respectively. Continue to incubate them in a cell incubator at 37 °C and 5% CO2 for 1 and 6 h respectively. Then, aspirate the culture medium containing the DLNPs@T@SIRT3 solution, wash the culture plates 3 times with PBS, and add 200 μL and 200 μM lysosomal green fluorescent probe LysoTracker Green respectively to stain the lysosomes for 2 h; after 2 h, wash the dishes 3 times with PBS, add 200 μL Hoechst dye solution respectively to stain the cell nuclei for 15 min, then wash the dishes 3 times with PBS, add 500 μL culture medium to each dish respectively, and evaluate the lysosomal escape of the lipid nanoparticles DLNPs@T@SIRT3 in cells at different time points under a confocal microscope.

[0062] The lysosomal escape effect of the lipid nanoparticles DLNPs@T@SIRT3 measured in this example in Raw 264.7 macrophages is as follows Figure 5 shown. In Raw 264.7 macrophages, when DLNPs@T@SIRT3 is administered for 6 h, it has a good lysosomal escape effect.

[0063] Example 6 Preparation method, morphology and particle size evaluation of mitochondrial-derived vesicles MDVs (SIRT3+) and

[0064] mitochondrial-derived vesicles MDVs (SIRT3+)Preparation method: Prepare lipid nanoparticles DLNPs@T@SIRT3 according to Example 1. Culture Raw 264.7 macrophages in a 10-cm-diameter culture dish with a culture medium volume of 7 mL. Add IL-4 to Raw 264.7 macrophages at a volume ratio of IL-4 solution: culture medium of 1:1000 to induce them into a senescent state for 24 h. Add the final nanopreparation DLNPs@T@SIRT3 to the senescent Raw 264.7 macrophages. Specifically, the mass of the SIRT3 plasmid is 15 μg, and continue to culture for 24 h after administration to enable the senescent Raw 264.7 macrophages to recover their functions under the action of SIRT3 and nanoparticles, and update the senescent state. The updated macrophages first extract mitochondria through a mitochondrial extraction kit (KeyGen Biotech; Cat.NO:KGA3106-50). Incubate the mitochondria with the supernatant in the last step of mitochondrial extraction at 37 °C for 2 h, and finally ultracentrifuge at 120,000 g for 2 h. The resulting precipitate is the mitochondrial-derived vesicles MDVs (SIRT3+) 。

[0065] Mitochondrial-derived vesicles MDVs (SIRT3+) Evaluate its morphology and particle size through TEM and Malvern particle size analyzer. Finally, it is shown that the mitochondrial-derived vesicles MDVs (SIRT3+) are round and cup-shaped, and the particle size distribution is uniform, which is beneficial for efficient cell uptake, as Figure 6 shown

[0066] Mitochondrial-derived vesicles MDVs (SIRT3-) Preparation method: Extract mitochondria from senescent Raw 264.7 macrophages through a mitochondrial extraction kit. Incubate the mitochondria with the supernatant in the last step of mitochondrial extraction at 37 °C for 2 h, and finally ultracentrifuge at 120,000 g for 2 h. The resulting precipitate is the mitochondrial-derived vesicles MDVs (SIRT3-) 。

[0067] Preparation method of mitochondrial-derived vesicles ΔMDVs: Extract mitochondria from normal Raw 264.7 through a mitochondrial extraction kit. Incubate the mitochondria with the supernatant in the last step of mitochondrial extraction at 37 °C for 2 h, and finally ultracentrifuge at 120,000 g for 2 h. The resulting precipitate is the mitochondrial-derived vesicles ΔMDVs

[0068] Example 7 Mitochondrial-derived vesicles MDVs (SIRT3+) Evaluation of the targeting effect on mitochondria in macrophages

[0069] Prepare mitochondrial-derived vesicles MDVs according to Example 6 (SIRT3+), and stained with DiI dye; Seeded Raw264.7 macrophages at 5×10 4 cells / well into 2 confocal dishes respectively, and after culturing in a cell incubator at 37°C and 5% CO2 for 24 h, when Raw 264.7 macrophages completely covered the confocal dishes, pour out the culture medium, wash the dishes 3 times with PBS, and then add mitochondrial-derived vesicles MDVs (SIRT3+) respectively. After continuing to incubate in a cell incubator at 37°C and 5% CO2 for 1 h and 4 h respectively, aspirate the culture medium containing the vesicles, wash the culture plates 3 times with PBS, and then add 200 μL of 100 μM mitochondrial green fluorescent probe Mito Tracker Green to stain the mitochondria. After staining for 30 min, wash the dishes 3 times with PBS, add 200 μL of Hoechst staining solution to stain the nuclei respectively, and after staining for 15 min, wash the dishes 3 times with PBS. Then add 500 μL of culture medium to each dish respectively, and evaluate the targeting effect of mitochondrial-derived vesicles MDVs (SIRT3+) on mitochondria in macrophages under a confocal microscope.

[0070] The targeting effect of mitochondrial-derived vesicles MDVs (SIRT3+) measured in this example on Raw 264.7 macrophages is as Figure 7 shown. In Raw 264.7 macrophages, when mitochondrial-derived vesicles MDVs (SIRT3+) are administered for 4 h, they have a good mitochondrial targeting effect.

[0071] Example 8 Evaluation of the regulation of mitochondrial-derived vesicles MDVs (SIRT3+) on macrophages

[0072] Prepare mitochondrial-derived vesicles MDVs (SIRT3+) according to Example 6. Seed Raw 264.7 macrophages at 2×10 5 cells / well into a six-well plate, and after culturing in a cell incubator at 37°C and 5% CO2 for 24 h, when Raw 264.7 macrophages completely cover the cell plate, treat Raw264.7 macrophages with IL-4 solution for 24 h to create a cell senescence environment. After culturing for 24 h, add mitochondrial-derived vesicles MDVs (SIRT3+) and incubate under the culture conditions of 37°C and 5% CO2 for 24 h. Then aspirate the culture medium containing the vesicles, wash the culture plates 3 times with PBS, digest the cells with an appropriate amount of PBS solution, add lysis buffer to the digested cells for lysis, and after complete lysis, centrifuge and keep the supernatant for ELISA kit detection. Parallel use mitochondrial-derived vesicles MDVs (SIRT3-)and mitochondrial-derived vesicles ΔMDVs extracted from normal Raw 264.7 macrophages were used as controls.

[0073] The mitochondrial-derived vesicles MDVs measured in this example (SIRT3+) The regulatory effect on Raw 264.7 macrophages is as Figure 8 shown. When Raw 264.7 macrophages are in the senescent state, mitochondrial-derived vesicles MDVs (SIRT3+) can better restore the energy metabolism function in macrophages, renewing the senescent macrophages; mitochondrial-derived vesicles ΔMDVs extracted from normal Raw 264.7 macrophages also have the effect of regulating macrophage metabolism, while mitochondrial-derived vesicles MDVs extracted from senescent Raw 264.7 macrophages (SIRT3-) cannot restore the function of macrophages and cannot alleviate the senescence of macrophages. Mitochondrial-derived vesicles MDVs (SIRT3+) were evaluated by differentiating the regulatory effects on key substances in macrophage energy metabolism.

[0074] Example 9 Mitochondrial-derived vesicles MDVs (SIRT3+) Evaluation of the regulatory effect on myofibroblasts

[0075] Mitochondrial-derived vesicles MDVs were prepared according to the method of Example 6 (SIRT3-) , ΔMDVs, MDVs (SIRT3+) , L929 cells were seeded into the lower chamber of a transwell cell culture insert at a density of 3×10 4 cells / well. After 24 h, TGF-β was added to the L929 cells at a volume ratio of TGF-β solution: culture medium of 1:500 to induce them into myofibroblasts for 24 h; macrophages were pretreated with IL-4 solution for 24 h according to the method in Example 6, and the induced Raw 264.7 macrophages were seeded into the upper chamber of the transwell cell culture insert at a density of 1×10 4 cells / well, as shown in A in Figure 9 . After culturing for 24 h, mitochondrial-derived vesicles MDVs (SIRT3+) were added to the upper chamber cells and incubated for another 24 h under the culture conditions of 37 °C and 5% CO2.

[0076] Absorb the culture medium in the lower chamber, wash the culture plate three times with PBS, add the fixative. After 15 min, aspirate the fixative, wash the culture plate three times with PBS, add Collagen-I and α-SMA antibodies respectively, and incubate overnight at 4°C. After 12 h, aspirate the antibodies, wash the culture plate three times with PBS, add Cy3 antibody and incubate for 4 h. After 4 h, aspirate the antibody, wash the culture plate three times with PBS, add Hoechst stain to stain the cell nuclei, aspirate the stain after 15 min, wash the culture plate three times with PBS, and add 1 mL of PBS solution. Observe the expression of the two antibodies under a fluorescence microscope, using mitochondrial-derived vesicles MDVs extracted from senescent Raw 264.7 macrophages (SIRT3-) and mitochondrial-derived vesicles ΔMDVs extracted from normal Raw 264.7 macrophages as controls. The senescence group was the group induced by IL-4 but not treated. The results are as Figure 9 shown in C below.

[0077] Prepare mitochondrial-derived vesicles MDVs according to the preparation in Example 6 (SIRT3-) 、ΔMDVs、MDVs (SIRT3+) . Seed L929 cells into the lower chamber of a transwell cell culture insert at a density of 3×10 4 cells / well, and seed Raw 264.7 macrophages into the upper chamber of the transwell cell culture insert at a density of 1×10 4 cells / well. Pretreat L929 cells and Raw 264.7 macrophages separately with TGF-β solution and IL-4 solution for 24 h respectively. After culturing for 24 h, add mitochondrial-derived vesicles MDVs (SIRT3+) for treatment to the cells in the upper chamber, using mitochondrial-derived vesicles MDVs extracted from senescent Raw 264.7 macrophages (SIRT3-) and mitochondrial-derived vesicles ΔMDVs extracted from normal Raw 264.7 macrophages as controls. After 24 h, aspirate the culture medium in the lower chamber, wash the culture plate three times with PBS, add 1 mL of trypsin solution to digest the myofibroblasts. After 1 min, add PBS solution to digest the myofibroblasts, add lysis buffer and lyse for 30 min. Centrifuge the lysed suspension at 2000 g for 10 min, take the supernatant and detect the content of hydroxyproline through a kit to evaluate the regulatory effect of mitochondrial-derived vesicles MDVs (SIRT3+) on myofibroblasts.

[0078] The results are as Figure 9 shown in B below, showing that mitochondrial-derived vesicles MDVs (SIRT3+) can better alleviate the fibrosis condition, while mitochondrial-derived vesicles MDVs extracted from senescent Raw 264.7 macrophages (SIRT3-)The function of myofibroblasts cannot be restored, and it has no effect on reducing the degree of fibrosis. Mitochondrial-derived vesicles MDVs (SIRT3+) Evaluate the regulatory effect on myofibroblasts by discrimination.

[0079] Example 10 Mitochondrial-derived vesicles MDVs (SIRT3+) Evaluation of the accumulation of MDVs in the fibrotic site

[0080] Prepare mitochondrial-derived vesicles MDVs according to Example 6 (SIRT3+) , where MDVs (SIRT3+) are labeled with DiI, and MDVs (SIRT3+) are administered to bleomycin (BLM)-induced pulmonary fibrosis mice by tail vein injection, and the in vivo fluorescence distribution of the pulmonary fibrosis mice is observed at different times after tail vein administration. In addition, free DiI is administered to pulmonary fibrosis mice by tail vein injection as a control.

[0081] In this example, the accumulation of mitochondrial-derived vesicles MDVs (SIRT3+) in the fibrotic site is as Figure 10 shown. Mitochondrial-derived vesicles MDVs (SIRT3+) are effectively delivered to the fibrotic site and accumulate, and the accumulation time lasts up to 72 hours. No obvious fluorescence is found in the mice injected with only free DiI, indicating the accumulation of mitochondrial-derived vesicles MDVs (SIRT3+) .

[0082] Example 11 Mitochondrial-derived vesicles MDVs (SIRT3+) Evaluation of the therapeutic effect on pulmonary fibrosis mice

[0083] Treat pulmonary fibrosis mice by tail vein injection with MDVs (SIRT3+) , ΔMDVs, MDVs (SIRT3-) and lipid nanoparticles DLNPs@T@SIRT3 respectively, where the treatment strategy is to perform tail vein injection every three days for a total of 7 times. The duration from the start of administration to the end of the experiment is 21 days. After the experiment, the mice are euthanized, and the lung tissues of the mice are collected and fixed with 4% paraformaldehyde. Normal is the normal mouse control, and BLM is the blank control for pulmonary fibrosis mice.

[0084] In this example, the therapeutic effect of mitochondrial-derived vesicles MDVs (SIRT3+) on pulmonary fibrosis mice is judged by COX2 and VEGF staining of the pathological features of pulmonary fibrosis, as Figure 11 shown. In mitochondrial-derived vesicles MDVs (SIRT3+)Under the treatment, the pulmonary parenchymalization of the mice was less severe than that of the pulmonary fibrosis mice (BLM), and the numbers of COX2 and VEGF were close to the levels of normal mice (Normal), indicating that the mitochondrial-derived vesicles MDVs of the present invention (SIRT3+) can reverse the development of pulmonary fibrosis to a certain extent and have a therapeutic effect on pulmonary fibrosis.

[0085] According to the pathological conditions of the disease occurrence, the present invention designs the pharmaceutical preparation scheme in multiple aspects and verifies it. Finally, it is found that the mitochondrial-derived vesicles MDVs (SIRT3+) can achieve a precise targeted therapeutic effect on pulmonary fibrosis. In addition, the preparation process of the mitochondrial-derived vesicles is simple, and the materials are easy to obtain, having the potential for wide application.

[0086] The above specific embodiments do not constitute a limitation on the protection scope of the present invention. Those skilled in the art should understand that various modifications, combinations, sub-combinations and substitutions can be made according to design requirements and other factors. Any modifications, equivalent substitutions and improvements made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for preparing mitochondrial-derived vesicles, characterized in that, The method for preparing the mitochondrial-derived vesicles comprises the following steps: 1) Add the LNPs (lipid nanoparticles) into a triphenylphosphine solution. After mixing the SIRT3 plasmid and the lipid nanoparticle solution at a ratio of 1:20 to 1:40 for 1 - 2 h, add them into senescent macrophages and continue culturing for 24 h - 48 h; the triphenylphosphine solution is a mixed solution of triphenylphosphine, EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide). 2) Then extract the mitochondria from the macrophages, incubate the mitochondria with the supernatant in the last step of mitochondrial extraction at 37 °C for 2 - 4 hours, and finally obtain the mitochondrial-derived vesicles by ultracentrifugation.

2. The preparation method according to claim 1, characterized in that, The method for preparing the LNPs lipid nanoparticles comprises: dissolving the co-lipid, amphiphilic lecithin, cholesterol, DC cholesterol, and liposomal phospholipid in an organic solvent, and preparing the nanoparticles by the ethanol injection method.

3. The preparation method according to claim 2, wherein, The co-lipid is selected from DOPE (dioleoylphosphatidylethanolamine); the amphiphilic lecithin is selected from at least one of soybean lecithin, egg yolk lecithin, hydrogenated lecithin, deoxycholine, and isolecithin; the liposomal phospholipid is DSPE-xPEGy, where x is selected from at least one of -CH3, -NH2, -COOH, -MAL, -OCH3; and the molecular weight of y is selected from at least one of 1000, 2000, 5000.

4. The preparation method according to claim 2, characterized in that, The mass ratio of the co-lipid: amphiphilic lecithin: cholesterol: DC cholesterol: liposomal phospholipid CL is 2:8:2:6:

1.

5. The preparation method according to claim 1, wherein The organic solvent is selected from at least one of methanol, ethanol, chloroform, and dichloromethane.

6. The preparation method according to claim 1, characterized in that, The mass ratio of triphenylphosphine, EDC, and NHS is 4:5:

6.

7. The mitochondrial-derived vesicles obtained by the preparation method according to any one of claims 1 - 6.

8. A preparation comprising the mitochondrial-derived vesicles prepared by the preparation method according to any one of claims 1 - 6.

9. Use of the mitochondrial-derived vesicles according to claim 7 or the preparation according to claim 8 in the preparation of a drug for a disease related to abnormal expression of apoptosis genes.

10. The application according to claim 9, wherein The diseases include pulmonary fibrosis, tumors, inflammatory diseases, or metabolic diseases.

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