An engineered mitochondria, its preparation method and application

Abstract

This invention discloses an engineered mitochondria, its preparation method, and its application. The preparation method of the engineered mitochondria includes: constructing immortalized mesenchymal stem cells; isolating and extracting mitochondria from the immortalized mesenchymal stem cells; pretreating the immortalized mesenchymal stem cells with IFN-γ and TNF-α to induce apoptosis, and extracting engineered apoptotic extracellular vesicle membranes; mixing the mitochondria with the engineered apoptotic extracellular vesicle membranes, and obtaining engineered mitochondria with their surface coated with the engineered apoptotic extracellular vesicle membranes after sonication and centrifugation. This effectively improves the stability and targeting of the engineered mitochondria, not only solving the problem of low cell uptake efficiency during direct mitochondrial transplantation, but also enhancing the functional recovery effect of mitochondria in damaged cells, providing a more efficient and stable source of mitochondria for engineered mitochondrial transplantation therapy.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an engineered mitochondria, its preparation method, and its application. Background Technology

[0002] Stem cell therapy, also known as regenerative medicine, involves culturing and expanding healthy stem cells in vitro, followed by transplantation into the patient. Utilizing the homing ability, differentiation potential, and immunomodulatory properties of stem cells, it aims to repair and replace damaged cells or tissues, thereby improving disease conditions. Immortalized mesenchymal stem cells (IMSCs), as a specially treated type of stem cell, combine the multi-lineage differentiation potential and immortalization characteristics of IMSCs. They not only provide a stable cell resource but can also differentiate into various tissue cell types, demonstrating immense potential in tissue engineering and regenerative medicine, offering new possibilities for the treatment of complex diseases. However, despite the numerous advantages of IMSCs, stem cell therapy still faces the significant challenge of immune rejection in clinical applications. Traditional stem cell transplantation requires strict tissue matching to avoid strong immune rejection reactions, which greatly limits its clinical application.

[0003] To overcome these challenges, scientists have explored innovative pathways for stem cell therapy, with mitochondrial transplantation emerging as a promising frontier. Mitochondria, acting as the cell's "energy factories," are responsible for synthesizing adenosine triphosphate (ATP), providing essential energy for cells, and participating in a series of key physiological processes such as fatty acid metabolism, cellular calcium buffering, and reactive oxygen species production. Mitochondrial transplantation aims to restore mitochondrial function and enhance cell vitality in damaged cells by directly transplanting healthy exogenous mitochondria or indirectly transplanting them into damaged organisms using cells carrying mitochondria. It has demonstrated significant therapeutic effects in various disease areas, such as ischemic myocardial injury, neurodegenerative diseases, and metabolic disorders. However, mitochondrial transplantation also faces a series of technical challenges in clinical application. How to efficiently and stably obtain a sufficient number of mitochondria to meet treatment needs, and how to address the low cell uptake efficiency and poor targeting issues inherent in direct mitochondrial transplantation, are key problems that urgently need to be solved in the field of mitochondrial transplantation therapy. Summary of the Invention

[0004] This invention addresses the shortcomings of existing technologies by providing, on the one hand, an engineered mitochondria, its preparation method, and its application. The engineered mitochondria are coated with an engineered extracellular vesicle membrane of apoptotic cells, which not only solves the problem that traditional mitochondrial transplantation therapy cannot efficiently and stably obtain sufficient mitochondria, but also improves cellular uptake efficiency and targeting. On the other hand, this invention provides an application of engineered mitochondria.

[0005] To achieve the above objectives, 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. Mitochondria were isolated and extracted from immortalized mesenchymal stem cells;

[0009] S3. Immortalized mesenchymal stem cells were pretreated with IFN-γ and TNF-α, apoptosis was induced, and engineered apoptotic extracellular vesicle membranes were extracted.

[0010] S4. Mitochondria are mixed with engineered apoptotic extracellular vesicle membranes, and after sonication and centrifugation, engineered mitochondria with engineered apoptotic extracellular vesicle membranes on their surface are obtained.

[0011] Based on the above-mentioned technical means, by constructing immortalized mesenchymal stem cells and extracting mitochondria, and then coating the mitochondria with the extracellular vesicle membranes of apoptotic cells pretreated with IFN-γ and TNF-α, the stability and targeting of engineered mitochondria are effectively improved. This not only solves the problem of low cell uptake efficiency when mitochondria are directly transplanted, but also enhances the functional recovery effect of mitochondria in damaged cells, providing a more efficient and stable source of mitochondria for engineered mitochondrial transplantation therapy.

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

[0013] S11. Transducing hTERT and SV40LT genes into human umbilical cord-derived mesenchymal stem cells via lentiviral vectors.

[0014] S12, after being screened and expanded by G418, yielded immortalized mesenchymal stem cells with immortalization characteristics.

[0015] Based on the above-mentioned technical means, the immortalized mesenchymal stem cells obtained have special properties that show no signs of aging during long-term passage and can maintain mitochondrial donor function.

[0016] Furthermore, the method for extracting engineered apoptotic extracellular vesicle membranes in step S3 includes the following steps:

[0017] S31. Pretreatment: Immortalized mesenchymal stem cells were seeded in culture medium and pretreated with IFN-γ and TNF-α at a concentration of 20 ng / mL for 24 h.

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

[0019] S33. Centrifuge the supernatant 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 hours, and then subjected to sonication and centrifugation to obtain the engineered apoptotic extracellular vesicle membrane.

[0021] Furthermore, the centrifugation conditions described in step S33 are as follows:

[0022] The supernatant was first centrifuged at 800g for 10 min to collect supernatant two; supernatant two was centrifuged at 2000g for 10 min to collect supernatant three; supernatant three was centrifuged at 16000g for 30 min to obtain the precipitate, which was engineered apoptotic extracellular vesicles.

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

[0024] Furthermore, the ultrasonic treatment conditions in step S34 are: power 40W, pulse period 6s on, 3s off, duration 6min; the centrifugation conditions are: the ultrasonically treated solution is first centrifuged at 3200g for 5min, and the supernatant is collected; the supernatant is then centrifuged at 20000g for 20min to obtain the precipitate as engineered apoptotic extracellular vesicle membrane.

[0025] Furthermore, in step S4, the mass ratio of the mitochondria to the engineered apoptotic extracellular vesicle membrane is 1:1.

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

[0027] An engineered mitochondria prepared according to the above-described method for preparing engineered mitochondria.

[0028] Based on the aforementioned technical means, engineered mitochondria, centered on exogenous mitochondria, are coated with an engineered apoptotic extracellular vesicle membrane (EIMSCs-apoEVs membrane). This allows them to target macrophages at sites of inflammation and is rich in various inflammatory regulatory molecules and tissue repair-related functional proteins. It can promote the transformation of M1 macrophages into M2 phenotypes. Through the targeted delivery efficiency mediated by the EIMSCs-apoEVs membrane, the uptake rate of immortalized mesenchymal stem cell-derived mitochondria is improved, thus providing a new technical pathway for improving the efficiency of mitochondrial transplantation therapy.

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

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

[0031] The beneficial effects achieved by this invention are as follows:

[0032] 1. This invention constructs immortalized mesenchymal stem cells as the source of mitochondria. The stable in vitro proliferation capacity of immortalized mesenchymal stem cells not only ensures the sufficiency of mitochondria in quantity and the integrity of their function, but also, due to their immortalized nature, provides a stable supply of mitochondrial resources. This avoids the difficulties and limited quantities of mitochondrial extraction found in traditional methods, simplifies the mitochondrial acquisition process, and improves extraction efficiency. This lays a solid foundation for the large-scale clinical application of mitochondrial transplantation therapy. Simultaneously, pretreatment of immortalized mesenchymal stem cells with IFN-γ and TNF-α induces apoptosis, and the apoptotic extracellular vesicle membrane is extracted. This results in the apoptotic extracellular vesicle membrane possessing good biocompatibility and targeting properties, enabling it to serve as a "carrier" for mitochondria. This significantly improves the cell uptake efficiency and targeting of engineered mitochondria during transplantation, solving the key technical challenges of low cell uptake efficiency and difficulty in accurately locating damaged sites in traditional direct mitochondrial transplantation.

[0033] 2. In this invention, engineered mitochondria are created by encapsulating exogenous mitochondria with the extracellular vesicle membrane of apoptotic cells. This not only protects the structure and function of mitochondria from damage by the external environment but also enhances the stability and functional recovery ability of mitochondria within damaged cells. The engineered extracellular vesicle membrane of apoptotic cells has the ability to target macrophages at sites of inflammation and is rich in various inflammatory regulatory molecules and tissue repair-related functional proteins. This promotes the transformation of M1 macrophages to the M2 phenotype, enabling engineered mitochondria to more effectively restore mitochondrial function and enhance cell viability after transplantation into damaged organisms. This not only provides a new technical pathway 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. The method for preparing engineered mitochondria in this invention involves 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 to obtain engineered mitochondria that can significantly regulate the immune function and energy metabolism reprogramming of macrophages. The preparation method is simple, efficient, green and pollution-free, suitable for large-scale production, and has important clinical significance and broad market application prospects. Attached Figure Description

[0035] Figure 1 The image shows a fluorescence microscope image of the mitochondria of primary MSCs and IMSCs of this invention, stained with MitoTracker Deep Red.

[0036] Figure 2 This is a flow cytometry analysis of mitochondria in primary MSCs and IMSCs of this invention, using MitoTracker Deep Red staining.

[0037] Figure 3 This is a flow cytometry analysis of mitochondria in primary MSCs and IMSCs of this invention, labeled with MitoTracker Green staining.

[0038] Figure 4 The image shows the mitochondria of primary MSCs and IMSCs of this invention detected by a multi-functional microplate reader using MitoTracker Deep Red staining.

[0039] Figure 5 The image shows the mitochondria of primary MSCs and IMSCs of this invention detected by a multi-functional microplate reader using MitoTracker Green staining.

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

[0041] Figure 7 This is a transmission electron microscope image of IMSCs-derived mitochondria from this invention.

[0042] Figure 8 This is a diagram illustrating the expression analysis of key mitochondrial proteins derived from IMSCs in this invention.

[0043] Figure 9 This is a graph showing the ATP production capacity of IMSCs-derived mitochondria in this invention.

[0044] Figure 10 Characterization of the engineered mitochondrial ultrastructure of this invention;

[0045] Figure 11 This is a diagram illustrating the engineered mitochondrial internalization process of this invention.

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

[0047] Figure 13 This diagram illustrates the regulatory effect of engineered mitochondria on the expression level of pro-inflammatory factor mRNA in macrophages.

[0048] Figure 14 This diagram illustrates the regulatory effect of engineered mitochondria on the expression of pro-inflammatory cytokine proteins in macrophages according to the present invention.

[0049] Figure 15 This diagram illustrates the regulatory effect of engineered mitochondria on the expression of anti-inflammatory factors in macrophages according to the present invention.

[0050] Figure 16 This diagram illustrates the regulatory effect of engineered mitochondria on the expression of anti-inflammatory factor proteins in macrophages according to the present invention.

[0051] Figure 17 This is a diagram showing the effect of the engineered mitochondria of this invention on the level of reactive oxygen species (ROS) in macrophage mitochondria;

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

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

[0054] Figure 20 This image shows the therapeutic effect of the engineered mitochondria of this invention on a mouse model of collagen-induced arthritis (CIA).

[0055] Figure 21 This is a statistical chart of clinical arthritis in a mouse model of CIA treated with engineered mitochondria according to the present invention;

[0056] Figure 22 This is a section of proximal joint tissue from the engineered mitochondrial therapy mouse model for CIA of this invention;

[0057] Figure 23 This is a pathological analysis diagram of the distal joint tissue of the engineered mitochondrial therapy mouse model for CIA according to the present invention;

[0058] Figure 24 This is a histopathological analysis of the metatarsophalangeal joint tissue of the engineered mitochondrial therapy mouse model for CIA of the present invention.

[0059] Figure 25 This is a histopathological scoring diagram of the joint tissue of the engineered mitochondrial therapy mouse model for CIA according to the present invention;

[0060] Figure 26 This is a flowchart illustrating the preparation and application of the engineered mitochondria of this invention. Detailed Implementation

[0061] To better understand the technical solution, achieve the objectives, and beneficial effects of this invention, the following embodiments further illustrate the invention in detail. However, these embodiments should not be construed as limiting the invention. Non-essential improvements and adjustments made by those skilled in the art based on the above description are also considered to fall within the scope of protection of this invention. Experimental methods not specifying specific conditions in the embodiments are generally performed under conventional conditions or according to the conditions recommended by the reagent manufacturer. Unless otherwise specified, the reagents and equipment used in this invention are conventional reagents and equipment in this technical field.

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

[0063] Example 1: Construction of Immortalized Mesenchymal Stem Cells

[0064] Experimental methods

[0065] Specifically, the following steps are included:

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

[0067] 1) Preliminary preparations:

[0068] Plasmids were extracted using an endotoxin-free plasmid mini-extraction kit (Tiangen Biotech, DP118); HEK 293T cells were processed at a concentration of 6 × 10⁻⁶. 5 One cell / well was seeded into a six-well plate and cultured for 24 hours for later use.

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

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

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

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

[0073] 1) hUC-MSCs cells were loaded at 8×10 4 10 cells / well were seeded into a 12-well plate and cultured for 6 hours for later use;

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

[0075] 3) Replace with fresh culture medium and continue subculture for 4 days;

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

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

[0078] Example 2: Isolation and extraction of mitochondria from constructed immortalized mesenchymal stem cells

[0079] 2.1 The Mitochondria Isolation Kit for Cultured Cells (ThermoFisher Scientific, 89874) was used, such as... Figure 6 As shown, the specific steps are as follows:

[0080] 1) Pre-cool the cell homogenizer on ice and set aside;

[0081] 2) Take the immortalized mesenchymal stem cells obtained under the conditions of Example 1, and collect 2 × 10⁻⁶ cells. 7 The cell suspension was centrifuged at 850g for 2 minutes at 4°C, the supernatant was discarded, and the cell pellet was collected.

[0082] 3) Add 800 μL of pre-cooled mitochondrial separation reagent A to the cell pellet, vortex to mix for 5 seconds, and then incubate on ice for 2 minutes.

[0083] 4) Transfer the cell suspension after ice bath incubation to a pre-cooled cell homogenizer and homogenize the cells under ice bath conditions;

[0084] 5) Transfer the completely lysed cell suspension to a centrifuge tube, add 800 μL of mitochondrial separation reagent C; rinse the grinder with 200 μL of mitochondrial separation reagent A, collect the rinsing solution and add it to 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 12000g for 15min to separate and obtain mitochondrial precipitate;

[0086] 7) Add 500 μL of mitochondrial separation reagent C to the mitochondrial precipitate for resuspending, and then centrifuge at 12000g for 5 min; the final precipitate obtained is IMSCs-derived mitochondria (Mito), which should be stored on ice for later use.

[0087] 2.2 Identification and Analysis

[0088] 1) The obtained IMSCs-derived mitochondria were morphologically observed using a transmission electron microscope;

[0089] The results are as follows Figure 7 As shown, mitochondria with intact structure, distinct morphological features, and a particle size of 200 nm were observed, proving that the mitochondria in IMSCs maintained good structural integrity during the isolation process.

[0090] 2) Western blot analysis:

[0091] Western blotting was used: mitochondrial precipitate derived from IMSCs was taken, 100 μL of 2% CHAPS was added and vortexed for 1 min to obtain mitochondrial samples;

[0092] Primary mesenchymal stem cells (MSCs) were lysed with RIPA; after lysis, the cells were sonicated on ice, and then the sonicated solution was centrifuged at 12,000 rpm for 10 min at 4 °C to obtain MSCs samples.

[0093] The proteins in mitochondrial and MSC samples were quantified using the BCA protein quantification kit, and the protein concentration was adjusted according to the quantification results. After mixing, 5× loading buffer was added, and the mixture was treated in a boiling water bath for 5-10 minutes to denature the proteins.

[0094] After separation by SDS-PAGE electrophoresis, the processed samples were transferred to a polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was then blocked with 5% skim milk (prepared with TBST solution) for 60 min. It was then incubated overnight at 4 °C with the corresponding primary antibody. After washing, it was incubated with HRP-labeled secondary antibody at room temperature for 1 h. Finally, the protein bands were observed and recorded using the Immobilon Western Chemilum HRP substrate for color development and the ChemiDocXRS blot imaging system.

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

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

[0097] ATP content determination: 100 μL of IMSCs-derived mitochondrial suspension was added to each well of an opaque 96-well plate, ensuring each well contained culture medium. Control wells were also included, containing heat-inactivated IMSCs-derived mitochondrial suspension. At room temperature, an equal volume of ATP was added to each well. The reagents were in the same volume as the cell culture medium. The contents of each well were mixed for 2 minutes using a fixed-track shaker to induce cell lysis. The cells were then incubated at room temperature for 10 minutes. The luminescence signal of each well was recorded, and the integration reading time was set to 0.25-1 second.

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

[0099] Example 3: Extraction of engineered extracellular vesicle membranes from apoptotic cells

[0100] 3.1 Apoptosis Induction

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

[0102] 2) After pretreatment with IFN-γ and TNF-α, the cells were washed twice with PBS, then replaced with basal medium without cell additives, and 500 nM astrococcus was added to induce apoptosis. After 16 h of apoptosis induction, the supernatant was collected.

[0103] 3.2 Extraction of engineered apoptotic extracellular vesicles

[0104] The collected supernatant was centrifuged at 800g for 10 minutes to obtain supernatant II.

[0105] Centrifuge supernatant two at 2000g for 10 minutes to collect supernatant three;

[0106] The supernatant was centrifuged at 16000g for 30 minutes, and the precipitate was collected.

[0107] The precipitate was resuspended in PBS to obtain engineered apoptotic extracellular vesicles;

[0108] 3.3 Extraction of extracellular vesicle membranes from engineered apoptotic cells

[0109] The extracted engineered apoptotic extracellular vesicles were resuspended in 0.25×PBS at 4℃ and allowed to stand for 2 hours. Then, they were treated with ultrasound using a probe. The ultrasound treatment conditions were 40W power, pulse period of 6s on and 3s off, and duration of 6min.

[0110] The sonicated solution was centrifuged at 3200g for 5 minutes at 4°C to obtain supernatant IV. Supernatant IV was centrifuged at 20000g for 20 minutes, the supernatant was discarded, and the collected precipitate was resuspended in 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] like Figure 26 As shown:

[0113] Mitochondria derived from IMSCs obtained under the conditions of Example 2 and engineered apoptotic extracellular vesicle membranes obtained under the conditions of Example 3 were mixed in a 1:1 mass ratio and then subjected to sonication at 4°C with a sonication power of 40W, a pulse cycle of 2s on and 3s off, and a duration of 2min to obtain a sonicated mixture.

[0114] The ultrasonic mixture was centrifuged at 12000g for 5 minutes at 4°C. The supernatant was discarded and the precipitate was collected as engineered mitochondria (eMito).

[0115] The engineered mitochondria were obtained and their morphology was observed using a transmission electron microscope.

[0116] The results are as follows Figure 10 As shown, the ultrastructural characterization of engineered mitochondria is a typical double-membrane structure, that is, engineered apoptotic extracellular vesicle membranes cover the mitochondrial surface to form a complete structure with a particle size of 500 nm.

[0117] Example 5: Determination of mitochondrial content and function

[0118] 1. Experimental materials

[0119] Primary mesenchymal stem cells (MSCs) and IMSCs obtained under the conditions of Example 1.

[0120] 2. Experimental Methods

[0121] MitoTracker staining treatment:

[0122] MSCs and IMSCs, or mitochondria isolated from them, were suspended in 500 μL of serum-free medium, and 300 nM MitoTracker Deep Red or MitoTracker Green fluorescent dye were added, respectively. The cells were stained at 37°C for 20 min. After staining, the staining working solution was discarded, and the cells were washed twice with PBS and then resuspended in fresh medium. Finally, the expression level of fluorescence signal was detected by flow cytometry, fluorescence microscopy, and multi-functional microplate reader.

[0123] 3. Experimental Results

[0124] Observation of mitochondria stained with MitoTracker Deep Red under a fluorescence microscope (e.g.) Figure 1As shown), the fluorescence intensity of mitochondria labeled with MitoTracker Deep Red and MitoTracker Green was detected by flow cytometry (e.g., as shown). Figure 2 and Figure 3 As shown in the figure, this demonstrates that both IMSCs and MSCs contain abundant functional mitochondria;

[0125] Furthermore, the fluorescence intensity of mitochondria stained with MitoTracker Deep Red and MitoTracker Green was detected using a multi-functional microplate reader (e.g., ...). Figure 4 and Figure 5 Quantitative analysis (as shown) revealed that, under the same cell number conditions, the fluorescence intensity of MitoTracker Deep Red and MitoTracker Green in the IMSCs group was significantly higher than that in the MSCs group, indicating that IMSCs have higher mitochondrial content and stronger mitochondrial functional activity.

[0126] In summary, IMSCs not only have a high mitochondrial content, but also exhibit superior mitochondrial functional characteristics, making them an ideal cell source for mitochondrial transplantation therapy.

[0127] Example 6: The 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) Suspend macrophage cells or mitochondria isolated from IMSCs in 500 μL of serum-free culture medium, add 300 nM MitoTracker Deep Red or MitoTracker Green fluorescent dye respectively, and stain at 37°C for 20 min.

[0132] 2) After staining, discard the staining working solution, wash twice with PBS, and then resuspend in fresh culture medium;

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

[0134] 4) After co-incubation, remove the cell supernatant and wash the cells twice with PBS;

[0135] 5) Cells were fixed with 4% paraformaldehyde, and macrophage nuclei were localized by 4',6-diamidinyl-2-phenylindole DAPI staining;

[0136] 6) Immunofluorescence microscopy was used to observe the uptake of engineered mitochondria by macrophages. The engineered mitochondria were stained with MitoTracker Deep Red fluorescence and then co-incubated with macrophages. The fluorescence intensity was detected by a multi-functional microplate reader.

[0137] 3. Experimental Results

[0138] like Figure 11 As shown, macrophage nuclei stained blue with DAPI, engineered mitochondria stained green with MitoTracker Green, and mitochondria stained red with MitoTracker Deep Red, indicating that the engineered mitochondria were successfully internalized into the macrophage cytoplasm. Specifically, in vitro co-culture experiments confirmed that macrophages can efficiently take up eMito, and eMito and the mitochondria of the recipient macrophages showed yellow fluorescence co-localization.

[0139] like Figure 12 As shown, engineered apoptotic extracellular vesicle membranes can promote the uptake of exogenous mitochondria by macrophages and significantly improve 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] like Figure 26 As 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 mitochondrial groups, and engineered apoptotic extracellular vesicle membranes were added to M1 and M2 macrophages, respectively, to a final concentration of 12.5 μg / mL, and incubated for 18 h.

[0147] 3) After 18 hours, cells were 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). At the same time, enzyme-linked immunosorbent assay (ELISA) was used to detect the protein expression of related cytokines (IL-6, TNF-α, IL-1β, TGF-β and PD-L1 mRNA) in the cell supernatant.

[0148] 3. Experimental Results

[0149] like Figure 13 As shown, the expression levels of pro-inflammatory cytokines IL-6, TNF-α, and IL-1β in macrophages were detected by real-time quantitative qPCR. eMito transplantation significantly inhibited the 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 IMSCs-derived mitochondrial group or the engineered apoptotic extracellular vesicle membrane group.

[0150] like Figure 14 As shown, the 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 cell 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] like Figure 15 As shown, the expression levels of the anti-inflammatory factors TGF-β and PD-L1 in macrophages by eMito 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 all significantly upregulated the mRNA expression levels of TGF-β and PD-L1, among which the upregulation effect of the eMito transplantation group was the most significant.

[0152] like Figure 16 As shown, the expression level of TGF-β protein, an anti-inflammatory factor in macrophages, was detected by enzyme-linked immunosorbent assay (ELISA). 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 cell extracellular vesicle membrane group.

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

[0154] Example 8: Energy metabolism of macrophages by engineered mitochondria

[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 levels using MitoSOX fluorescent probe

[0159] The MitoSOX reagent stock solution was diluted with PBS at a ratio of 1:10000 to prepare a working solution with a final concentration of 500 nM. 1-2 mL of the freshly prepared MitoSOX working solution was added to each well. The cells were incubated at 37°C with 5% CO2 for 30 min. After incubation, the cells were washed with PBS, and this process was repeated three times. Finally, the samples were analyzed using a multi-functional fluorescent microplate reader.

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

[0161] Cells were suspended in 0.5 mL of complete culture medium, and an equal volume of JC-1 staining working solution was added. After gentle inversion and mixing, the cells were incubated 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-chilled JC-1 staining buffer: first, the cells were resuspended in 1 mL of buffer, centrifuged at 600 g for 4 min at 4 °C and the supernatant was discarded; then, the cells were resuspended in JC-1 staining buffer and the fluorescence intensity was measured using a multi-functional microplate reader.

[0162] 3) ATP production level

[0163] Prepare an opaque 96-well plate. Add 100 μL of eMito, IMSCs-derived mitochondrial matrix, and engineered apoptotic extracellular vesicle membrane suspension to each well, ensuring each well contains culture medium. Include control wells containing culture medium but without cells or mitochondria. Equilibrate the plate and its samples to room temperature. Add an equal volume of [unspecified substance] to each well. The reagents were prepared in the same volume as the cell culture medium. The contents of each well were mixed for 2 minutes using a fixed-track shaker to induce cell lysis. The plates were incubated at room temperature for 10 minutes. Finally, the luminescence signal of each well was recorded, and the reading time was set to 0.25-1 second.

[0164] 3. Experimental Results

[0165] 1) Results are as follows Figure 17 As shown, fluorescence intensity was detected by a multi-functional microplate reader, revealing that mitochondrial transplantation significantly reduced ROS levels in macrophages; and compared to the Mito transplantation group, the eMito transplantation group showed a more significant trend of ROS level downregulation, demonstrating that eMito has a superior biological effect in regulating the oxidative stress balance of macrophages.

[0166] 2) Results are as follows Figure 18As shown, fluorescence intensity was detected by a multi-functional microplate reader. Compared with the PBS control group, the Mito group, and the engineered apoptotic extracellular vesicle membrane group, macrophages in the eMito transplantation group showed 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 mitochondrial function of macrophages, promote the recovery of damaged mitochondrial membrane potential, and thus maintain the integrity of mitochondrial structure.

[0167] 3) Results as follows Figure 19 As shown, fluorescence intensity was detected by a multi-functional microplate reader, revealing that the eMito transplantation group significantly enhanced the ATP production capacity of pro-inflammatory macrophages compared to the PBS control group.

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

[0169] Example 9: In vivo experiments on engineered mitochondria

[0170] 1. Experimental materials

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

[0172] 2. Experimental Methods

[0173] A CIA mouse model was established. Mitochondrial transplantation was performed on all CIA model mice 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. The treatment was administered on day 27 after immunization. The above treatments were given via tail vein injection every other day for a total of 3 treatments. During the experiment, the weight changes of each group of animals were monitored and recorded daily, the degree of paw swelling was measured, and the clinical symptom scores of the limbs were recorded.

[0174] 3. Experimental Results

[0175] like Figure 20 As shown, the joints of CIA mice were photographed and compared on day 50. Compared with the normal control group, the mice in the PBS treatment group showed significant foot inflammation, characterized by swelling in the ankle joint area, thickening of the foot pad, and deformity of the interphalangeal joints. The eMito treatment group showed the most significant effect, with only mild redness and swelling of 1-2 toe joints and the foot edema basically disappearing. The improvement of clinical symptoms was close to that of the healthy control group.

[0176] like Figure 21 As shown, the mice were grossly assessed every other day, and the clinical scores of arthritis were summarized. It was found that although Mito alone and engineered apoptotic extracellular vesicle membranes showed certain disease-relieving effects through clinical scores of experimental animals in each group, the eMito intervention group had a significant advantage in terms of treatment effect.

[0177] In summary, eMito has significant advantages in relieving joint inflammation;

[0178] Mice were humanely euthanized on day 50, and their joints were rapidly dissected and fixed overnight with 4% paraformaldehyde. After decalcification, the joints were embedded in paraffin and sectioned. Sections were stained with H&E, and all sections were imaged using a NanoZoomer S360 digital slide scanner. Figure 22-24 As shown, the results indicated that, compared with the control group and the other two treatment groups, the CIA model animals that received eMito transplantation exhibited significant pathological improvements; specifically, the degree of synovial inflammation was reduced, the integrity of the joint structure was maintained, and the destruction of cartilage tissue was reduced.

[0179] In addition, the severity of articular cartilage damage was graded from 0 (no damage) to 4 (severe damage), with two volunteers scoring and the results tallied. Figure 25 As shown, no experimental animals died or experienced significant adverse reactions during the entire experimental period in the eMito treatment group, indicating that the treatment regimen has good safety.

[0180] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method of preparing an engineered mitochondrion, characterized by, Comprise the following steps: S1, constructing immortalized mesenchymal stem cells; S2, separating and extracting mitochondria from the immortalized mesenchymal stem cells; S3, taking the immortalized mesenchymal stem cells, pre-treating with IFN-γ and TNF-α, apoptosis induction, and extracting to obtain the engineered apoptotic extracellular vesicle membrane; S4, mixing the mitochondria with the engineered apoptotic extracellular vesicle membrane, and obtaining the engineered mitochondria coated with the engineered apoptotic extracellular vesicle membrane after ultrasonic treatment and centrifugation; The construction of the 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 through a lentivirus vector; S12, obtaining immortalized mesenchymal stem cells with immortalization characteristics after G418 screening and amplification culture; The extraction method of the engineered apoptotic extracellular vesicle membrane in step S3 comprises the following steps: S31, pre-treatment: taking the immortalized mesenchymal stem cells, inoculating in the culture medium, and pre-treating with IFN-γ at a concentration of 20 ng / mL and TNF-α at a concentration of 20 ng / mL for 24 h; S32, apoptosis induction: inducing apoptosis of the cells pre-treated with IFN-γ and TNF-α for 16 h using staurosporine, and collecting the supernatant; S33, centrifuging the supernatant to obtain the engineered apoptotic extracellular vesicle; S34, resuspending the engineered apoptotic extracellular vesicle in PBS at 4°C, standing for 2 h, and obtaining the engineered apoptotic extracellular vesicle membrane after ultrasonic treatment and centrifugation.

2. The method of claim 1, wherein the engineered mitochondria are prepared by the method comprising the steps of: The centrifugation condition in step S33 is: The supernatant is first centrifuged at 800 g for 10 min, and the second supernatant is collected; the second supernatant is centrifuged at 2000 g for 10 min, and the third supernatant is collected; the third supernatant is centrifuged at 16000 g for 30 min, and the precipitate is the engineered apoptotic extracellular vesicle.

3. The method for preparing engineered mitochondria according to claim 1, characterized in that, The ultrasonic treatment condition in step S34 is: power 40W, pulse cycle on 6s and off 3s, and duration 6min; the centrifugation condition is: the ultrasonic treated solution is first centrifuged at 3200 g for 5 min, and the fourth supernatant is collected; the fourth supernatant is centrifuged at 20000 g for 20 min, and the precipitate is the engineered apoptotic extracellular vesicle membrane.

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

1.

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

6. The engineered mitochondria prepared by the preparation method of the engineered mitochondria according to any one of claims 1-5.

7. The engineered mitochondria of claim 6, wherein, The particle size of the engineered mitochondria is 200-500 nm.

8. The engineered mitochondria according to claim 6 for use in the preparation of a medicament for treating arthritis.

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