Engineered mitochondria as well as preparation method and application thereof

Abstract

The invention discloses engineered mitochondria as well as a preparation method and application thereof. The preparation method of the engineered mitochondria comprises the following steps: constructing immortalized mesenchymal stem cells; separating and extracting mitochondria from the immortalized mesenchymal stem cells; taking the immortalized mesenchymal stem cells, carrying out IFN-gamma and TNF-alpha pretreatment on the immortalized mesenchymal stem cells, carrying out apoptosis induction, and extracting to obtain an engineered apoptotic extracellular vesicle membrane; mitochondria and the engineered apoptotic extracellular vesicle membrane are mixed, and after ultrasonic treatment and centrifugation, engineered mitochondria with the engineered apoptotic extracellular vesicle membrane coated on the surface is obtained; the engineering mitochondria has the advantages that the stability and the targeting property of the engineering mitochondria are effectively improved, the problem of low cell uptake efficiency during direct transplantation of the mitochondria is solved, the function recovery effect of the mitochondria in damaged cells is enhanced, and a more efficient and stable mitochondria source is provided for transplantation treatment of the engineering mitochondria.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to an engineered mitochondria and a preparation method and application thereof. Background Art

[0002] Stem cell therapy, also known as regenerative medicine, involves amplifying healthy stem cells through in vitro culture and then transplanting them into the patient's body. It uses the homing effect, differentiation potential and immunomodulatory properties of stem cells to repair and replace damaged cells or tissues, thereby improving the disease state. Among them, immortalized mesenchymal stem cells (IMSCs) are a type of specially treated stem cells that combine the multidirectional differentiation potential and immortality of mesenchymal stem cells. They can not only provide stable cell resources, but also differentiate into a variety of tissue cells. They show great potential in the fields of tissue engineering and regenerative medicine, and provide new possibilities for the treatment of complex diseases. However, despite the many advantages of IMSCs, stem cell therapy still faces a major challenge in clinical application: immune rejection. Traditional stem cell transplantation requires strict tissue matching to avoid strong immune rejection reactions, which greatly limits its scope of clinical application.

[0003] In order to overcome the above problems, scientists have explored innovative paths for stem cell therapy, among which mitochondrial transplantation therapy has become a highly anticipated frontier direction. Mitochondria, as the "energy factory" in cells, are responsible for synthesizing adenosine triphosphate (ATP), providing cells with necessary energy support, and participating in a series of key physiological processes such as fatty acid metabolism, cellular calcium buffering, and reactive oxygen production. Mitochondrial transplantation therapy aims to restore the mitochondrial function of damaged cells and enhance cell vitality by directly transplanting healthy exogenous mitochondria or indirectly transplanting them into the damaged body with the help of cells carrying mitochondria. It has shown significant therapeutic effects in multiple disease treatment areas, such as myocardial ischemic injury, neurodegenerative diseases, and metabolic diseases. However, mitochondrial transplantation therapy also faces a series of technical difficulties in clinical applications. How to efficiently and stably obtain a sufficient number of mitochondria to meet treatment needs, and how to solve the problems of low cell uptake efficiency and poor targeting in direct mitochondrial transplantation are key issues that need to be urgently addressed in the current field of mitochondrial transplantation therapy. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention provides, on the one hand, an engineered mitochondria and a preparation method and application thereof. The surface of the engineered mitochondria is coated with an engineered apoptotic cell extracellular vesicle membrane, which not only solves the problem that traditional mitochondrial transplantation therapy in the prior art cannot efficiently and stably obtain sufficient mitochondria, but also improves the cellular uptake efficiency and targeting; on the other hand, provides an application of the engineered mitochondria.

[0005] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] A method for preparing engineered mitochondria, comprising the following steps:

[0007] S1. Construct immortalized mesenchymal stem cells;

[0008] S2. Isolate and extract mitochondria from the immortalized mesenchymal stem cells;

[0009] S3. Take the immortalized mesenchymal stem cells pretreated with IFN-γ and TNF-α, induce apoptosis, and extract the engineered apoptotic extracellular vesicle membrane;

[0010] S4. Mix the mitochondria with the engineered apoptotic extracellular vesicle membrane, and after ultrasonic treatment and centrifugation, obtain engineered mitochondria with the surface coated with the engineered apoptotic extracellular vesicle membrane.

[0011] According to the above technical means, by constructing immortalized mesenchymal stem cells and extracting mitochondria, and combining the apoptotic extracellular vesicle membrane pretreated with IFN-γ and TNF-α to coat the mitochondria, the stability and targeting of the engineered mitochondria are effectively improved. It not only solves the problem of low cell uptake efficiency during direct transplantation of mitochondria, but also enhances the functional recovery effect of mitochondria in damaged cells, providing a more efficient and stable mitochondrial source for engineered mitochondrial transplantation therapy.

[0012] Further, the construction of the immortalized mesenchymal stem cells in step S1 includes the following steps:

[0013] S11. Transduce the hTERT and SV40LT genes into human umbilical cord-derived mesenchymal stem cells through a lentiviral vector;

[0014] S12. After screening with G418 and amplification culture, obtain immortalized mesenchymal stem cells with immortalized characteristics.

[0015] According to the above technical means, the obtained immortalized mesenchymal stem cells with immortalized characteristics have no signs of senescence during long-term passage due to their special properties, and can maintain the mitochondrial donor function.

[0016] Further, the extraction method of the engineered apoptotic extracellular vesicle membrane in step S3 includes the following steps:

[0017] S31. Pretreatment: Take the immortalized mesenchymal stem cells and inoculate them in a culture medium, and pretreat them with IFN-γ at a concentration of 20 ng / mL and TNF-α at a concentration of 20 ng / mL for 24 h;

[0018] S32. Apoptosis induction: The cells pretreated with IFN-γ and TNF-α were induced to apoptosis by staurosporine for 16 h, and the supernatant was collected.

[0019] S33. The supernatant was centrifuged to obtain engineered apoptotic extracellular vesicles.

[0020] S34. The engineered apoptotic extracellular vesicles were resuspended in PBS at 4°C, allowed to stand for 2 h, and then after ultrasonic treatment and centrifugation, the membrane of the engineered apoptotic extracellular vesicles was obtained.

[0021] Further, the centrifugation conditions in step S33 were as follows:

[0022] The supernatant was first centrifuged at 800 g for 10 min to collect the second supernatant; the second supernatant was centrifuged at 2000 g for 10 min to collect the third supernatant; the third supernatant was centrifuged at 16000 g for 30 min, and the precipitate obtained was the engineered apoptotic extracellular vesicles.

[0023] According to the above technical means, centrifugation at 800 g for 10 min was used to remove cell debris in the supernatant; centrifugation at 2000 g for 10 min was used to remove large particle impurities in the second supernatant; centrifugation at 16000 g for 30 min, and the precipitate obtained was resuspended in PBS to obtain engineered apoptotic extracellular vesicles (EIMSCs-apoEVs).

[0024] Further, the ultrasonic treatment conditions in step S34 were: power 40 W, pulse cycle on for 6 s, off for 3 s, and duration 6 min; the centrifugation conditions were: the solution after ultrasonic treatment was first centrifuged at 3200 g for 5 min to collect the fourth supernatant; the fourth supernatant was centrifuged at 20000 g for 20 min, and the precipitate obtained was the membrane of the engineered apoptotic extracellular vesicles.

[0025] Further, the mass ratio of the mitochondria to the membrane of the engineered apoptotic extracellular vesicles in step S4 was 1:1.

[0026] Further, the ultrasonic treatment conditions in step S4 were: power 40 W, pulse cycle on for 2 s, off for 3 s, and duration 2 min; the centrifugation conditions were: centrifugation at 12000 g for 5 min.

[0027] An engineered mitochondrion prepared by the method for preparing the above-mentioned engineered mitochondrion.

[0028] According to the above technical means, the engineered mitochondria are centered around exogenous mitochondria, and their surfaces are coated with engineered apoptotic extracellular vesicle membranes (EIMSCs-apoEVs membranes), which have the ability to target macrophages at the inflammatory site and are rich in various inflammation-regulating related molecules and tissue repair-related functional proteins. They can promote the transformation of M1 macrophages into the M2 phenotype, and through the targeted delivery efficiency mediated by the EIMSCs-apoEVs membrane, the cellular uptake rate of immortalized mesenchymal stem cell-derived mitochondria is increased, thus providing a new technical path for improving the efficiency of mitochondrial transplantation therapy.

[0029] Furthermore, the particle size of the engineered mitochondria is 200-500 nm.

[0030] Furthermore, the application of the engineered mitochondria in the preparation of drugs for regulating macrophage immunity and metabolism.

[0031] The beneficial effects achieved by the present invention:

[0032] 1. By constructing immortalized mesenchymal stem cells as the source of mitochondria, the present invention not only ensures the sufficiency of mitochondria in quantity and the integrity of their functions under the stable in vitro proliferation ability of immortalized mesenchymal stem cells, but also enables the stable supply of mitochondrial resources due to the immortalized characteristics, avoiding the problems of difficult mitochondrial extraction and limited quantity in traditional methods, simplifying the process of mitochondrial acquisition, and improving the extraction efficiency of mitochondria, laying a solid foundation for the large-scale clinical application of mitochondrial transplantation therapy. At the same time, by pretreating immortalized mesenchymal stem cells with IFN-γ and TNF-α, inducing their apoptosis and extracting apoptotic extracellular vesicle membranes, the apoptotic extracellular vesicle membranes have good biocompatibility and targeting ability, can serve as a "carrier" for mitochondria, significantly improve the cellular uptake efficiency and targeting of engineered mitochondria during transplantation, and solve the key technical problems of low cellular uptake efficiency and difficulty in accurately locating the damaged site during traditional direct mitochondrial transplantation.

[0033] 2. In the present invention, the engineered mitochondria are coated with exogenous mitochondria by combining apoptotic extracellular vesicle membranes, which not only protects the structure and function of mitochondria from damage by the external environment, but also enhances the stability and function recovery ability of mitochondria in damaged cells. The engineered apoptotic extracellular vesicle membranes have the ability to target macrophages at the inflammatory site and are rich in various inflammation-regulating related molecules and tissue repair-related functional proteins. Under the action of promoting the transformation of M1 macrophages into the M2 phenotype, the engineered mitochondria can more effectively restore the mitochondrial function of damaged cells and enhance cell viability after transplantation into the damaged body. It not only provides a new technical path for mitochondrial transplantation therapy, but also brings new hope for the treatment of refractory diseases and is expected to play an important role in future clinical practice.

[0034] 3. In the present invention, the preparation method of engineered mitochondria obtains engineered mitochondria that can significantly regulate the immune function and energy metabolism reprogramming of macrophages through simple and easy-to-operate steps such as mixing mitochondria extracted from immortalized mesenchymal stem cells with engineered apoptotic extracellular vesicle membranes, sonication, and centrifugation. The preparation method is simple, highly efficient, green and pollution-free, suitable for large-scale production, and has important clinical significance and broad market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 It is a fluorescence microscope image of mitochondria of primary MSCs and IMSCs of the present invention stained with MitoTracker Deep Red;

[0036] Figure 2 It is a flow cytometry analysis diagram of mitochondria of primary MSCs and IMSCs of the present invention stained with MitoTracker Deep Red;

[0037] Figure 3 It is a flow cytometry analysis diagram of mitochondria of primary MSCs and IMSCs of the present invention stained with MitoTracker Green;

[0038] Figure 4 It is a detection diagram of a multifunctional microplate reader of mitochondria of primary MSCs and IMSCs of the present invention stained with MitoTracker Deep Red;

[0039] Figure 5 It is a detection diagram of a multifunctional microplate reader of mitochondria of primary MSCs and IMSCs of the present invention stained with MitoTracker Green;

[0040] Figure 6 It is a schematic diagram of the extraction process of IMSCs-derived mitochondria of the present invention;

[0041] Figure 7 It is a transmission electron microscope image of IMSCs-derived mitochondria of the present invention;

[0042] Figure 8 It is an analysis diagram of the expression of key proteins of IMSCs-derived mitochondria of the present invention;

[0043] Figure 9 It is a determination diagram of the ATP generation ability of IMSCs-derived mitochondria of the present invention;

[0044] Figure 10 It is the ultrastructure characterization of the engineered mitochondria of the present invention;

[0045] Figure 11 This is the process diagram of the engineered mitochondrial internalization of the present invention;

[0046] Figure 12 This is the quantitative analysis diagram of the uptake efficiency of engineered mitochondria by macrophages of the present invention;

[0047] Figure 13 This is the regulation diagram of the expression level of pro-inflammatory factor mRNA in macrophages by engineered mitochondria of the present invention;

[0048] Figure 14 This is the regulation diagram of the protein expression of pro-inflammatory factors in macrophages by engineered mitochondria of the present invention;

[0049] Figure 15 This is the regulation diagram of the expression of anti-inflammatory factors in macrophages by engineered mitochondria of the present invention;

[0050] Figure 16 This is the regulation diagram of the protein expression of anti-inflammatory factors in macrophages by engineered mitochondria of the present invention;

[0051] Figure 17 This is the diagram of the effect of engineered mitochondria on the level of mitochondrial reactive oxygen species (ROS) in macrophages of the present invention;

[0052] Figure 18 This is the diagram of the effect of engineered mitochondria on the mitochondrial membrane potential of macrophages of the present invention;

[0053] Figure 19 This is the diagram of the effect of engineered mitochondria on ATP production in macrophages of the present invention;

[0054] Figure 20 This is the treatment effect diagram of engineered mitochondria on the collagen-induced arthritis (CIA) mouse model of the present invention;

[0055] Figure 21 This is the clinical arthritis statistical chart of the treatment of CIA mouse model with engineered mitochondria of the present invention;

[0056] Figure 22 This is the proximal joint tissue section diagram of the treatment of CIA mouse model with engineered mitochondria of the present invention;

[0057] Figure 23 This is the distal joint histopathological analysis diagram of the treatment of CIA mouse model with engineered mitochondria of the present invention;

[0058] Figure 24 This is the histopathological analysis diagram of the metatarsophalangeal joint of the treatment of CIA mouse model with engineered mitochondria of the present invention;

[0059] Figure 25 This is the joint tissue pathological score diagram of the treatment of CIA mouse model with engineered mitochondria of the present invention;

[0060] Figure 26 This is the flow chart for the preparation and application of the engineered mitochondria of the present invention. Detailed implementation manners

[0061] To better understand the technical solution, objectives and beneficial effects of the present invention, the present invention is further specifically described through the following embodiments. However, it should not be construed as a limitation to the present invention. For those skilled in the art, some non-essential improvements and adjustments made according to the above invention content are also considered to fall within the protection scope of the present invention. The experimental methods without specific conditions noted in the embodiments are usually carried out under conventional conditions or according to the conditions recommended by the reagent manufacturers. Unless otherwise specified, the reagents and equipment used in the present invention are conventional reagents and equipment in the technical field.

[0062] Unless otherwise specified, the reagents and materials used in the following embodiments are all commercially available.

[0063] Example 1: Construction of immortalized mesenchymal stem cells

[0064] Experimental method

[0065] Specifically, it includes the following steps:

[0066] 1.1 Construction of lentiviral vectors of human telomerase reverse transcriptase (hTERT) and simian virus 40 large T antigen (SV40LT):

[0067] 1) Preliminary preparation:

[0068] Use an endotoxin-free plasmid midiprep kit (Tiangen Biotech, DP118) to extract plasmids; inoculate HEK 293T cells at 6×10 5 cells / well in a six-well plate and culture for 24 h for standby;

[0069] Transfection reagent Lipofectamine TM LTX Reagent with PLUS TM Reagent (purchased from Invitrogen);

[0070] 2) Add 150 μL of Opti-MEM medium into two 1.5 mL centrifuge tubes respectively. Add psPAX2, pMD2.G and the target gene vector plasmid into one centrifuge tube with a mass ratio of 1.5:0.5:2. Then add 4 μL of PLUS reagent and mix well. Label it as tube A. Add 12 μL of transfection reagent into the other centrifuge tube and mix well. Label it as tube B. Add tube B into tube A and mix evenly. Let it stand for 5 min at room temperature.

[0071] 3) Slowly add the A + B mixture dropwise to HEK 293T cells for transfection. After 6 h of transfection, change the medium and replace the supernatant containing the transfection reagent with DMEM complete medium (containing 10% fetal bovine serum). After continuing transfection for 48 h, collect the supernatant of HEK293T cells, transfer it into a 15 mL centrifuge tube, and centrifuge at 2000 g for 5 min to remove cell debris and impurities to obtain lentivirus. Aliquot the lentivirus and store it at -80 °C for later use.

[0072] 1.2 Lentiviral transduction of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs cells) with hTERT and SV40LT:

[0073] 1) Seed hUC-MSCs cells at 8×10 4 cells / well into a 12-well plate and culture for 6 h for later use.

[0074] 2) After the cells adhere, add large T lentivirus at a ratio of 10 MOI and immediately add 40 μL of lentivirus enhanced infection solution P (HitransG P), and culture for 6 h. Then add large TERT lentivirus at a ratio of 10 MOI and transfect for 12 h.

[0075] 3) Replace with fresh medium and continue passage culture for 4 days.

[0076] 4) Replace with fresh medium and add 100 μg / mL of Geneticin (G418) for screening, and culture for 4 days.

[0077] 5) Replace with fresh medium and continue passage culture for 15 days. Change the medium every two days and passage if necessary. Finally, obtain immortalized mesenchymal stem cells (IMSCs), expand the cells, and freeze the cells in a liquid nitrogen tank for later use.

[0078] Example 2: Isolate and extract mitochondria from the constructed immortalized mesenchymal stem cells

[0079] 2.1 Use the Mitochondria Isolation Kit for Cultured Cells kit (ThermoFisher Scientific, 89874), as Figure 6 shown, the specific steps are as follows:

[0080] 1) Pre-cool the cell grinder on ice for later use;

[0081] 2) Take the immortalized mesenchymal stem cells obtained under the conditions of Example 1, collect 2×10 7 cell suspensions, centrifuge at 850 g for 2 min at 4 °C, discard the supernatant, and collect the cell pellet;

[0082] 3) Add 800 μL of pre-cooled mitochondrial isolation reagent A to the cell pellet, vortex for 5 s and then incubate on ice for 2 min;

[0083] 4) Transfer the cell suspension after ice bath incubation to a pre-cooled cell grinder and perform cell homogenization under ice bath conditions;

[0084] 5) Transfer the completely lysed cell suspension to a centrifuge tube, add 800 μL of mitochondrial isolation reagent C; and rinse the grinder with 200 μL of mitochondrial isolation reagent A, collect the rinse solution and transfer it into the centrifuge tube, gently invert and mix; then centrifuge at 700 g for 10 min at 4 °C and collect the supernatant;

[0085] 6) Centrifuge the collected supernatant at 12000 g for 15 min to separate the mitochondrial pellet;

[0086] 7) Resuspend the mitochondrial pellet with 500 μL of mitochondrial isolation reagent C, and then centrifuge at 12000 g for 5 min; the finally obtained pellet is IMSCs-derived mitochondria (mitochondria, Mito), which is stored on ice for later use.

[0087] 2.2 Identification and analysis

[0088] 1) Morphologically observe the obtained IMSCs-derived mitochondria by transmission electron microscopy;

[0089] The results are as Figure 7 shown. Mitochondria with intact structures, obvious morphological characteristics and a particle size of 200 nm are observed, which proves that the mitochondria in IMSCs maintain good structural integrity during the separation process;

[0090] 2) Western blot analysis:

[0091] Western blotting: Take the IMSCs-derived mitochondrial precipitate, add 100 μL of 2% CHAPS and vortex for 1 min to obtain mitochondrial samples;

[0092] Lyse primary mesenchymal stem cells (MSCs) by adding RIPA; after lysis, perform sonication on ice, and then centrifuge the sonicated solution at 12,000 rpm for 10 min at 4 °C to obtain MSCs samples;

[0093] Quantify the proteins in the mitochondrial samples and MSCs samples using a BCA protein quantification kit, and adjust the protein concentration according to the quantification results; after mixing, add 5× loading buffer and treat in a boiling water bath for 5 - 10 minutes to denature the proteins;

[0094] After separating the treated samples by SDS-PAGE electrophoresis, transfer them to a polyvinylidene difluoride membrane; then block the PVDF membrane with 5% skim milk (prepared with TBST solution) for 60 min; then incubate with the corresponding primary antibody overnight at 4 °C; after washing, incubate with the HRP-labeled secondary antibody for 1 h at room temperature; finally, develop color using Immobilon Western Chemilum HRP substrate, and observe and record the protein bands using the blot imaging system ChemiDoc XRS;

[0095] The results are as Figure 8 shown. The cytoplasmic marker β-actin was not detected in the IMSCs-derived mitochondria, while the mitochondrial-specific marker cytochrome c oxidase IV (CoxIV) showed significantly high expression, demonstrating that the obtained IMSCs-derived mitochondria had high purity and low cytoplasmic component contamination;

[0096] 3) Quantitative analysis of mitochondrial functional activity:

[0097] ATP content determination: Add 100 μL of the IMSCs-derived mitochondrial suspension to each well of a non-opaque 96-well plate, and ensure that each well contains culture medium. At the same time, set control wells with heat-inactivated IMSCs-derived mitochondria; at room temperature, add an equal volume of reagent, with the same volume as the cell culture medium; use an orbital shaker to mix the contents of each well for 2 min to promote cell lysis; then incubate at room temperature for 10 min; record the luminescence signal of each well, and set the integration reading time to 0.25 - 1 second;

[0098] The results are as Figure 9As shown, the ATP level of IMSCs-derived mitochondria was significantly higher than that of the control group, demonstrating that the IMSCs-derived mitochondria isolated by the experimental method of this study maintained intact biological activity, providing reliable experimental materials for subsequent related research.

[0099] Example 3: Extraction of Engineered Apoptotic Extracellular Vesicle Membranes

[0100] 3.1 Apoptosis Induction

[0101] 1) The IMSCs obtained under the conditions of Example 1 were inoculated into the culture medium. When the cell confluence reached 40%-50%, IFN-γ at a concentration of 20 ng / mL and TNF-α at a concentration of 20 ng / mL were added for pretreatment for 24 h.

[0102] 2) The cells pretreated with IFN-γ and TNF-α were washed twice with PBS, then changed to a basal medium without cell additives, and 500 nM staurosporine was added for apoptosis induction. After 16 h of apoptosis induction, the supernatant was collected.

[0103] 3.2 Extraction of Engineered Apoptotic Extracellular Vesicles

[0104] The above-collected supernatant was centrifuged at 800 g for 10 min to collect supernatant II.

[0105] Supernatant II was centrifuged at 2000 g for 10 min to collect supernatant III.

[0106] Supernatant III was centrifuged at 16000 g for 30 min to collect the precipitate.

[0107] The precipitate was resuspended with PBS to obtain engineered apoptotic extracellular vesicles.

[0108] 3.3 Extraction of Engineered Apoptotic Extracellular Vesicle Membranes

[0109] The extracted engineered apoptotic extracellular vesicles were resuspended with 0.25×PBS at 4°C, allowed to stand for 2 h, and then treated with probe ultrasound. The ultrasound treatment conditions were a power of 40 W, a pulse cycle of 6 s on and 3 s off, and a duration of 6 min.

[0110] The solution treated with ultrasound was centrifuged at 3200 g for 5 min at 4°C to collect supernatant IV; supernatant IV was centrifuged at 20000 g for 20 min, the supernatant was discarded, and the collected precipitate was resuspended with PBS to obtain engineered apoptotic extracellular vesicle membranes, which were stored at -80°C for later use.

[0111] Example 4: Preparation of Engineered Mitochondria

[0112] As Figure 26 shown:

[0113] Take the IMSCs-derived mitochondria obtained under the conditions of Example 2 and the engineered apoptotic extracellular vesicle membrane obtained under the conditions of Example 3, mix them at a mass ratio of 1:1, and then perform ultrasonic treatment at 4°C. The ultrasonic power is 40W, the pulse cycle is on for 2s and off for 3s, and the duration is 2min to obtain an ultrasonic mixture;

[0114] Centrifuge the ultrasonic mixture at 12,000g for 5min at 4°C, discard the supernatant, and collect the precipitate, which is the engineered mitochondria (Engineered mitochondria, eMito);

[0115] Perform morphological observation on the obtained engineered mitochondria using a transmission electron microscope;

[0116] The results are as Figure 10 shown. The ultrastructure of the engineered mitochondria is characterized by a typical double-membrane structure, that is, the engineered apoptotic extracellular vesicle membrane coats the surface of the mitochondria to form a complete structure, and its particle size is 500nm.

[0117] Example 5: Determination of Mitochondrial Content and Function

[0118] 1. Experimental Materials

[0119] Primary mesenchymal stem cells (Mesenchymal Stem Cells, MSCs), IMSCs obtained under the conditions of Example 1.

[0120] 2. Experimental Methods

[0121] MitoTracker staining treatment:

[0122] Suspend MSCs and IMSCs cells or mitochondria isolated and extracted from them in 500 μL of serum-free medium, add 300 nM of MitoTracker Deep Red or MitoTracker Green fluorescent dye respectively, and place them in an environment of 37°C for 20 min for staining; after staining is completed, discard the staining working solution, wash twice with PBS, and then resuspend with fresh medium; finally, analyze the expression level of the fluorescence signal by flow cytometry, fluorescence microscopy and a multifunctional microplate reader respectively.

[0123] 3. Experimental Results

[0124] By observing the mitochondria stained with MitoTracker Deep Red under a fluorescence microscope (as Figure 1As shown in the figure, the fluorescence intensity of mitochondria stained with MitoTracker Deep Red and MitoTracker Green was detected by flow cytometry (as Figure 2 and Figure 3 shown), which proved that both IMSCs and MSCs contained abundant functional mitochondria;

[0125] Furthermore, the fluorescence intensity of mitochondria stained with MitoTracker Deep Red and MitoTracker Green was detected by a multifunctional microplate reader (as Figure 4 and Figure 5 shown) for quantitative analysis. It was found that under the condition of the same cell number, the fluorescence intensities of MitoTracker Deep Red and MitoTracker Green in the IMSCs group were significantly higher than those in the MSCs group, indicating that IMSCs had a higher mitochondrial content and stronger mitochondrial functional activity;

[0126] In summary, IMSCs not only had a high mitochondrial content but also exhibited more excellent mitochondrial functional characteristics, making them an ideal cell source for mitochondrial transplantation therapy.

[0127] Example 6: Internalization process of engineered mitochondria

[0128] 1. Experimental materials

[0129] eMito and macrophages obtained under the conditions of Example 4.

[0130] 2. Test methods

[0131] 1) Macrophages or mitochondria isolated from IMSCs were suspended in 500 μL of serum-free medium, and 300 nM of MitoTracker Deep Red or MitoTracker Green fluorescent dye was added respectively, and then incubated at 37 °C for 20 min;

[0132] 2) After staining, the staining working solution was discarded, and the cells were washed twice with PBS and then resuspended in fresh medium;

[0133] 3) The stained macrophages were co-incubated with eMito, and a control group was set up simultaneously;

[0134] 4) After co-incubation, the cell supernatant was removed, and the cells were washed twice with PBS;

[0135] 5) The cells were fixed with 4% paraformaldehyde, and the macrophage nuclei were located by staining with 4',6-diamidino-2-phenylindole DAPI;

[0136] 6) The uptake of engineered mitochondria by macrophages was observed using immunofluorescence microscopy. The engineered mitochondria were co-incubated with macrophages after being stained with MitoTracker Deep Red fluorescence, and a multifunctional microplate reader was used to detect the fluorescence intensity.

[0137] 3. Experimental results

[0138] As Figure 11 shown, the macrophage nuclei were stained blue with DAPI, the engineered mitochondria were stained green with MitoTracker Green, and the mitochondria were stained red with MitoTracker Deep Red, indicating that the engineered mitochondria were successfully internalized into the macrophage cytoplasm; specifically, through in vitro co-culture experiments, it was confirmed that macrophages could efficiently uptake eMito, and eMito showed yellow fluorescence co-localization with the mitochondria of recipient macrophages;

[0139] As Figure 12 shown, the membrane of engineered apoptotic extracellular vesicles could promote the uptake of exogenous mitochondria by macrophages, significantly increasing the internalization rate of exogenous mitochondria.

[0140] Example 7: Immunomodulation of macrophages by engineered mitochondria

[0141] 1. Experimental materials

[0142] eMito and macrophages obtained under the conditions of Example 4.

[0143] 2. Experimental methods

[0144] As Figure 26 shown:

[0145] 1) In this study, LPS combined with IFN-γ was used as an inducer of M1 macrophages, and IL-4 was used as an inducer of M2 macrophages to establish a macrophage polarization model;

[0146] 2) eMito, IMSCs-derived mitochondria, and the membrane of engineered apoptotic extracellular vesicles were respectively added to M1 and M2 macrophages at a final concentration of 12.5 μg / mL and incubated for 18 h;

[0147] 3) After 18 h, each group of cells was collected for qPCR detection to analyze the mRNA expression levels of inflammation-related cytokines (IL-6, TNF-α, IL-1β, TGF-β, and PD-L1 mRNA), and the protein expression of related cytokines (IL-6, TNF-α, IL-1β, TGF-β, and PD-L1 mRNA) in the cell supernatant was detected by enzyme-linked immunosorbent assay.

[0148] 3. Experimental Results

[0149] As Figure 13 shown, the mRNA expression levels of pro-inflammatory factors IL-6, TNF-α, and IL-1β in macrophages were detected by real-time quantitative qPCR. eMito transplantation could significantly inhibit the mRNA expression levels of pro-inflammatory cytokines IL-6, TNF-α, and IL-1β in M1 macrophages, and its inhibitory effect was significantly better than that of the mitochondrial group derived from IMSCs or the engineered apoptotic extracellular vesicle membrane group;

[0150] As Figure 14 shown, the protein expression levels of pro-inflammatory factors IL-6, TNF-α, and IL-1β in macrophages were detected by enzyme-linked immunosorbent assay. It was found that compared with the mitochondrial group and the engineered apoptotic extracellular vesicle membrane group, the eMito transplantation group had a more significant inhibitory effect on the secretion of IL-6 and TNF-α proteins by M1 macrophages;

[0151] As Figure 15 shown, the mRNA expression levels of anti-inflammatory factors TGF-β and PD-L1 in macrophages were detected by qPCR. In the study of M2 macrophages, compared with the PBS control group, the Mito group, the engineered apoptotic extracellular vesicle membrane group, and the eMito transplantation group could all significantly up-regulate the mRNA expression levels of TGF-β and PDL-1, and the up-regulation effect of the eMito transplantation group was the most significant;

[0152] As Figure 16 shown, the protein expression level of the anti-inflammatory factor TGF-β in macrophages was detected by enzyme-linked immunosorbent assay. It was found that the secretion level of TGF-β protein in the cell supernatant of the eMito transplantation group was significantly higher than that of the PBS control group, the Mito group, and the engineered apoptotic extracellular vesicle membrane group;

[0153] In summary, eMito transplantation can significantly regulate the inflammatory function of macrophages, and its immunomodulatory effect is significantly better than that of Mito and the engineered apoptotic extracellular vesicle membrane, proving that eMito transplantation may play an important role in immunomodulation by regulating macrophage function.

[0154] Example 8: Energy Metabolism of Engineered Mitochondria in Macrophages

[0155] 1. Experimental Materials

[0156] eMito and macrophages obtained under the conditions of Example 4.

[0157] 2. Experimental Methods

[0158] 1) Detection of Mitochondrial ROS Level by MitoSOX Fluorescent Probe

[0159] The stock solution of MitoSOX reagent was diluted with PBS at a dilution ratio of 1:10,000 to finally prepare a working solution with a concentration of 500 nM; 1 - 2 mL of the freshly prepared MitoSOX reagent working solution was added to each culture well; the cells were incubated in an incubator at 37°C and 5% CO2 for 30 min; after incubation, the cells were washed with PBS, and this process was repeated 3 times; finally, the samples were detected and analyzed using a multifunctional fluorescence microplate reader;

[0160] 2) Evaluation of mitochondrial membrane potential by JC-1 fluorescent probe staining

[0161] The cells were suspended in 0.5 mL of complete medium, and an equal volume of JC-1 staining working solution was added. After gently inverting and mixing, the cells were incubated in an incubator at 37°C for 20 min; after incubation, the cells were centrifuged at 600 g for 5 min at 4°C, and the supernatant was discarded; the cells were washed twice with pre-cooled JC-1 staining buffer: first, 1 mL of buffer was added to resuspend the cells, and the cells were centrifuged at 4°C and 600 g for 4 min, and the supernatant was discarded; then the cells were resuspended with JC-1 staining buffer, and the fluorescence intensity was measured using a multifunctional microplate reader;

[0162] 3) ATP production level

[0163] Prepare an opaque 96-well plate, add 100 μL of eMito, IMSCs-derived mitochondrial group, and engineered apoptotic extracellular vesicle membrane suspension to each well, and ensure that each well contains medium. At the same time, set up control wells containing medium but no cells or mitochondria; equilibrate the plate and the samples in it to room temperature; add an equal volume of reagent, with the same volume as the cell medium; use an orbital shaker to mix the contents of each well for 2 min to promote cell lysis; incubate the plate at room temperature for 10 min; finally, record the luminescence signal of each well, and set the integration reading time to 0.25 - 1 second.

[0164] 3. Experimental results

[0165] 1) The results were as Figure 17 shown. By detecting the fluorescence intensity using a multifunctional microplate reader, it was found that mitochondrial transplantation treatment could significantly reduce the ROS level in macrophages; and compared with the Mito transplantation group, the eMito transplantation group showed a more significant downward trend in ROS level, proving that eMito has better biological effects in regulating the oxidative stress balance of macrophages;

[0166] 2) The results were as Figure 18As shown in the figure, by detecting the fluorescence intensity with a multifunctional microplate reader, it was found that compared with the PBS control group, the Mito group, and the engineered apoptotic extracellular vesicle membrane group, macrophages in the eMito transplantation group showed a significantly enhanced fluorescence signal intensity in JC-1 staining. Specifically, the ratio of red fluorescence (representing polymers) to green fluorescence (representing monomers) in the eMito transplantation group was significantly increased, indicating that eMito transplantation can effectively improve the mitochondrial function of macrophages, promote the recovery of damaged mitochondrial membrane potential, and thus maintain the integrity of mitochondrial structure;

[0167] 3) The results are as Figure 19 shown in the figure. By detecting the fluorescence intensity with a multifunctional microplate reader, it was found that compared with the PBS control group, the eMito transplantation group significantly enhanced the ATP production ability of pro-inflammatory macrophages;

[0168] In summary, eMito transplantation has a significant regulatory effect on the energy metabolism reprogramming of macrophages. It supports the maintenance of macrophage function through specific molecular mechanisms and promotes the phenotypic polarization stability of macrophages in a specific microenvironment.

[0169] Example 9: In Vivo Experiment of Engineered Mitochondria

[0170] 1. Experimental Materials

[0171] eMito obtained under the conditions of Example 4, DBA / 1 mice (establishing CIA mouse models).

[0172] 2. Experimental Methods

[0173] CIA mouse models were established. All CIA model mice received mitochondrial transplantation treatment after the onset of arthritis. The animals were randomly divided into 5 groups: normal control group, PBS control group, Mito transplantation group, engineered apoptotic extracellular vesicle membrane treatment group, and eMito treatment group, on the 27th day after immunization. The above treatments were administered by tail vein injection, with a treatment frequency of once every other day for a total of 3 treatments. During the experiment, the body weight changes of animals in each group were monitored and recorded daily, the degree of paw swelling was detected, and the clinical symptom scores of the four limbs were recorded simultaneously.

[0174] 3. Experimental Results

[0175] As Figure 20 shown in the figure, on the 50th day, the joints of CIA mice were photographed and analyzed. Compared with the normal control group, mice in the PBS treatment group showed significant foot inflammation reactions, characterized by swelling in the ankle joint area, thickening of the footpad, and deformation of the interphalangeal joints. Among them, the eMito treatment group had the most significant effect, showing only slight redness and swelling of 1 - 2 toe joints, and the foot edema was basically disappeared. The improvement degree of its clinical symptoms was close to that of the healthy control group;

[0176] As Figure 21 shown, each mouse was grossly evaluated every other day, and the clinical scores of arthritis were summarized. It was found that although certain disease remission effects were shown by clinical scores of each group of experimental animals, with Mito alone and engineered apoptotic extracellular vesicles both showing some disease remission, the eMito intervention group had a significant advantage in terms of treatment effect;

[0177] In summary, eMito has a significant advantage in alleviating joint inflammation;

[0178] On the 50th day, the mice were humanely sacrificed, and the joints were quickly isolated and fixed in 4% paraformaldehyde overnight. After decalcification, they were paraffin-embedded and sectioned. The sections were stained with H&E, and all sections were imaged using a NanoZoomer S360 digital slide scanner. As Figures 22 - 24 shown, the results showed that compared with the control group and the other two treatment groups, the CIA model animals receiving eMito transplantation showed significant pathological improvement; specifically, it was manifested as a reduction in synovial inflammation, maintenance of joint structural integrity, and reduction in cartilage tissue damage;

[0179] In addition, the severity of joint cartilage damage was graded from 0 (no damage) to 4 (severe damage), and two volunteers scored and statistically analyzed it. As Figure 25 shown, during the entire experimental period, no experimental animals died or showed obvious adverse reactions in the eMito treatment group, indicating that this treatment plan has good safety.

[0180] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A method for preparing engineered mitochondria, characterized in that: The following steps are involved: S1. Construction of immortalized mesenchymal stem cells; S2. Isolation and extraction of mitochondria from immortalized mesenchymal stem cells; S3, immortalized mesenchymal stem cells were pretreated with IFN-γ and TNF-α, apoptosis was induced, and engineered apoptotic cell extracellular vesicle membranes were extracted; S4. The mitochondria are mixed with the engineered apoptotic cell extracellular vesicle membranes, and after sonication and centrifugation, engineered mitochondria with the engineered apoptotic cell extracellular vesicle membranes coated on the surface are obtained.

2. The method for preparing engineered mitochondria according to claim 1, characterized in that: The construction of immortalized mesenchymal stem cells in step S1 comprises the following steps: S11, transducing hTERT and SV40LT genes into human umbilical cord-derived mesenchymal stem cells via lentiviral vectors; S12. After G418 screening and expansion culture, immortalized mesenchymal stem cells with immortalization characteristics are obtained.

3. The method for preparing engineered mitochondria according to claim 1, characterized in that: The method for extracting the engineered apoptotic cell extracellular vesicle membrane in step S3 comprises the following steps: S31, pretreatment: immortalized mesenchymal stem cells were inoculated in culture medium and pretreated with 20 ng / mL IFN-γ and 20 ng / mL TNF-α for 24 h; S32, apoptosis induction: cells pretreated with IFN-γ and TNF-α were induced to apoptosis with staurosporine for 16 h, and the supernatant was collected; S33, centrifuging the supernatant to obtain engineered apoptotic extracellular vesicles; S34. Take the engineered apoptotic cell extracellular vesicles and resuspend them with PBS at 4°C, let them stand for 2 hours, and then obtain the engineered apoptotic cell extracellular vesicle membranes after ultrasonic and centrifugation treatments.

4. The method for preparing engineered mitochondria according to claim 3, characterized in that: The centrifugal treatment conditions in step S33 are: The supernatant was first centrifuged at 800g for 10 min to collect supernatant 2; supernatant 2 was centrifuged at 2000g for 10 min to collect supernatant 3; supernatant 3 was centrifuged at 16000g for 30 min to obtain the precipitate which was the engineered apoptotic extracellular vesicles.

5. The method for preparing engineered mitochondria according to claim 3, characterized in that: The ultrasonic treatment conditions in step S34 are: power 40W, pulse cycle on 6s, off 3s, duration 6min; the centrifugal treatment conditions are: the ultrasonically treated solution is first centrifuged at 3200g for 5min, and the supernatant four is collected; the supernatant four is centrifuged at 20000g for 20min, and the precipitate obtained is the engineered apoptotic cell extracellular vesicle membrane.

6. The method for preparing engineered mitochondria according to claim 1, characterized in that: The mass ratio of the mitochondria and the engineered apoptotic cell extracellular vesicle membrane mixed in step S4 is 1:

1.

7. The method for preparing engineered mitochondria according to claim 1, characterized in that: The ultrasonic treatment conditions in step S4 are: power 40W, pulse cycle on 2s, off 3s, duration 2min; the centrifugal treatment conditions are: centrifugation at 12000g for 5min.

8. An engineered mitochondria prepared according to the method for preparing engineered mitochondria according to any one of claims 1 to 7.

9. The engineered mitochondria according to claim 8, characterized in that The particle size of the engineered mitochondria is 200-500 nm.

10. Use of the engineered mitochondria according to claim 8 in the preparation of a drug for regulating macrophage immunity and metabolism.

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