Hybrid vesicles targeting mitochondria, preparation method and use

Abstract

The present application relates to a hybrid vesicle targeting mitochondria, a preparation method and application, which comprises mixing extracellular vesicles and a loading material loaded in the mixed extracellular vesicles, the loading material comprising mitochondria extracted from bone marrow mesenchymal stem cells pretreated with urolithin A, and the mixed extracellular vesicles comprising bone marrow mesenchymal stem cell extracellular vesicles and endothelial cell extracellular vesicles. The present application combines the functions of targeted delivery of mitochondria and activation of mitochondrial autophagy. By utilizing the regenerative function of BVs and the vascular endothelial cell targeting ability of EVs, exogenous functional mitochondria are delivered to supplement the mitochondrial pool and restore cell energy supply, while the pretreatment of urolithin A enhances the mitochondrial reception signal, promotes the removal of intracellular damaged mitochondria by restoring the redox balance and calcium balance on the mitochondrial-related endoplasmic reticulum membrane, and relieves the endoplasmic reticulum stress.

Description

Technical Field

[0001] This application relates to the field of biomaterials technology, and in particular to a hybrid vesicle targeting mitochondria, its preparation method, and its application. Background Technology

[0002] Mitochondria are indispensable organelles in cells, responsible for coordinating cellular energy metabolism, redox balance, calcium signaling, and programmed cell death, thereby maintaining cellular homeostasis and tissue integrity. Disruption of mitochondrial homeostasis—the dynamic imbalance between normal and dysfunctional mitochondria—is a key pathological driver affecting various tissues, including the heart, kidneys, lungs, and skeletal muscle. This balance is primarily maintained through mitophagy—a compromised process of selective mitophagy—to sustain bioenergy metabolism and cellular activity.

[0003] Under physiological conditions, mitochondrial targeting is maintained through several highly coordinated pathways. The typical PINK1-Parkin axis selectively recognizes damaged mitochondria: PINK1 stabilizes on depolarized mitochondrial membranes and recruits Parkin, enabling it to generalize outer membrane proteins and driving the recruitment of autophagy transfer proteins such as p62, OPTN, and NDP52. Simultaneously, receptor-dependent BNIP3, NIX, and FUNDC1 respond to hypoxia or energy stimuli by directly binding to LC3 to promote mitochondrial autophagy formation. These pathways work together to ensure the continuous turnover of damaged mitochondria and contribute to maintaining mitochondrial homeostasis, energy metabolism, and cellular viability. Defects in mitophagy lead to dysfunctional mitochondrial accumulation, adenine triphosphate (ATP) depletion, and excessive production of mitochondrial reactive oxygen species (mtROS), all of which exacerbate oxidative stress and inflammation.

[0004] Furthermore, mitochondria are functionally and structurally bound to the endoplasmic reticulum (ER) via mitochondrial-associated endoplasmic reticulum membranes (MAMs), serving as a crucial hub for calcium and reactive oxygen species (ROS) metabolism. Under pathological conditions, excessive calcium influx from the ER to the mitochondria triggers mitochondrial depolarization and excessive ROS production, while mitochondrial dysfunction further exacerbates ER stress by hindering ATP-dependent protein folding and activating the unfolded protein response (UPR). This feedback loop creates a vicious cycle, where defects in mitophagy and persistent ER stress jointly exacerbate cellular damage. Increasing evidence suggests that the breakdown of the mitochondrial-ER regulatory axis is a common pathological mechanism in metabolic diseases, chronic wounds, neuroinflammatory diseases, and acute injuries. Therefore, the simultaneous regulation of mitochondrial autophagy and ER homeostasis is essential for restoring intracellular homeostasis. Summary of the Invention

[0005] This application provides a mitochondrial-targeting hybrid vesicle, its preparation method, and its application, which combines the functions of targeted delivery to mitochondria and activation of mitophagy, providing a comprehensive treatment option for diseases of mitochondrial dysfunction.

[0006] In a first aspect, embodiments of this application provide a mitochondrial-targeting hybrid vesicle, comprising a mixed extracellular vesicle and a loading material loaded in the mixed extracellular vesicle, the loading material comprising mitochondria extracted from bone marrow mesenchymal stem cells pretreated with urolithin A, the mixed extracellular vesicle comprising bone marrow mesenchymal stem cell extracellular vesicles and endothelial cell extracellular vesicles.

[0007] In conjunction with the first aspect, in one embodiment, the mass ratio of the bone marrow mesenchymal stem cell extracellular vesicles to the endothelial cell extracellular vesicles is (0.5-2):1.

[0008] In conjunction with the first aspect, in one embodiment, the mass ratio of the mixed extracellular vesicles to the loading material is in the range of 1:(2 to 4).

[0009] In conjunction with the first aspect, in one embodiment, the amount of urolithin A and the bone marrow mesenchymal stem cells treated with it is such that 8 mL of 4-8 μmol / L urolithin A is added to every 2 to 5 million bone marrow mesenchymal stem cells.

[0010] Secondly, embodiments of this application provide a method for preparing mitochondrial-targeting hybrid vesicles as described above, comprising: The extracellular vesicles of bone marrow mesenchymal stem cells and extracellular vesicles of endothelial cells were mixed and the mixed extracellular vesicles were extruded. A loading material comprising mitochondria was isolated from bone marrow mesenchymal stem cells pretreated with urolithin A; The loading material was mixed with hybrid extracellular vesicles by extrusion to obtain mitochondrial-targeted hybrid vesicles.

[0011] In conjunction with the second aspect, in one embodiment, the conditions for mixing the bone marrow mesenchymal stem cell extracellular vesicles and endothelial cell extracellular vesicles include: performing 4 to 6 cycles of ultrasound treatment in an ice-water bath, with each cycle of ultrasound treatment including an ultrasound amplitude of 20% to 40%, on for 20 to 40 seconds, and off for 1.5 to 2.5 minutes.

[0012] In conjunction with the second aspect, in one embodiment, a polycarbonate membrane with a pore size of 0.2 μm is extruded and the extrusion is repeated 10 times to obtain mixed extracellular vesicles.

[0013] In conjunction with the second aspect, in one embodiment, before mixing the loading material with the mixed extracellular vesicles, the preparation method further includes: incubating the mixed extracellular vesicles at 36.3°C to 37.2°C for 45 min to 75 min to promote membrane fusion.

[0014] In conjunction with the second aspect, in one embodiment, after mixing by extrusion, the preparation method further includes: incubating at 36.3℃~37.2℃ for 2 hours to obtain a crude product; centrifuging the crude product at 2000~3500×g at 4℃ for 45 minutes, followed by centrifugation at 4000~10000×g at 4℃ for 45 minutes; and then ultracentrifuging the resulting supernatant at 110000~200000×g at 4℃ for 70 minutes to collect nanoscale mitochondrial-targeting hybrid vesicles.

[0015] Thirdly, embodiments of this application provide the use of mitochondrial-targeting hybrid vesicles as described above in the preparation of medicaments for treating diabetic wounds or acute respiratory distress syndrome.

[0016] The beneficial effects of the technical solution provided in this application include: The mitochondrial-targeting hybrid vesicles UA-Mito@HEVs provided in this application serve as a highly efficient and biocompatible therapeutic platform, combining targeted mitochondrial delivery with activation of mitophagy. Utilizing the regenerative capabilities of vascular vesicles (BVs) and the vascular endothelial cell targeting ability of EVs, exogenous functional mitochondria are delivered to replenish the mitochondrial pool and restore cellular energy supply. Pretreatment with urolithin A enhances mitochondrial signal reception, promoting the clearance of intracellularly damaged mitochondria and alleviating endoplasmic reticulum stress by restoring redox and calcium balance on the mitochondrial-associated endoplasmic reticulum membrane. UA-Mito@HEVs offer a promising and translational strategy for addressing diseases caused by mitochondrial dysfunction and endoplasmic reticulum stress. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 The preparation and characterization results of UA-Mito@HEVs provided in the embodiments of this application; Figure 2 The embodiments of this application provide the results of UA-Mito@HEVs' internalization and targeted uptake in cells; Figure 3The in vitro efficacy results of UA-Mito@HEVs in promoting cell proliferation, migration and survival in an oxidative microenvironment provided in the embodiments of this application; Figure 4 The transcriptomic results of principal component analysis of HUVECs treated with UA-Mito@HEVs under oxidative stress conditions provided in the embodiments of this application; Figure 5 Transcriptomic analysis results of HUVECs treated with UA-Mito@HEVs under oxidative stress conditions for the present application regarding the processing of endoplasmic reticulum stress-related proteins in GSEA; Figure 6 Transcriptomic analysis results of HUVECs treated with UA-Mito@HEVs under oxidative stress conditions for the embodiments of this application regarding mitochondrial autophagy in GSEA animals; Figure 7 Transcriptomic analysis results of UA-Mito@HEVs treated with HUVECs under oxidative stress conditions for the present application embodiments regarding autophagy in animal cells of GSEA; Figure 8 Transcriptomic analysis results of autophagosome formation in GSEA of HUVECs treated with UA-Mito@HEVs under oxidative stress conditions provided in the embodiments of this application; Figure 9 Transcriptomic analysis results of UA-Mito@HEVs treated with HUVECs under oxidative stress conditions provided in this application embodiment regarding mitochondrial respiratory chain complex I in GSEA; Figure 10 The results of UA-Mito@HEVs improving mitochondrial bioenergetics under methylglyoxal (MGO) stress provided in the embodiments of this application; Figure 11 The results of UA-Mito@HEVs regulating mitophagy and endoplasmic reticulum-mitochondria interaction provided in the embodiments of this application; Figure 12 The UA-Mito@HEVs provided in the embodiments of this application significantly promote the repair of diabetic wounds; Figure 13 Histological and immunofluorescence analysis results of diabetic wound healing provided in the embodiments of this application; Figure 14 The UA-Mito@HEVs provided in the embodiments of this application significantly reduce lung injury in acute respiratory distress syndrome (ARDS). Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0020] Mitochondrial dysfunction leads to tissue damage in many diseases. Disruption of mitochondrial homeostasis results in the accumulation and insufficient clearance of dysfunctional mitochondria, hindering cellular energy metabolism and cell repair. Replenishing functional mitochondria while simultaneously clearing damaged mitochondria could be a promising approach to restoring mitochondrial homeostasis.

[0021] Based on this, embodiments of this application provide a hybrid vesicle targeting mitochondria, comprising a mixed extracellular vesicle and a loading material loaded in the mixed extracellular vesicle, the loading material comprising mitochondria extracted from bone marrow mesenchymal stem cells pretreated with urolithin A, the mixed extracellular vesicle comprising bone marrow mesenchymal stem cell extracellular vesicles and endothelial cell extracellular vesicles.

[0022] For ease of understanding, in this application, urolithiasis A is referred to as UA, mitochondria as Mito, mixed extracellular vesicles as HEVs, bone marrow mesenchymal stem cell extracellular vesicles as BVs, endothelial cell extracellular vesicles as EVs, and mitochondrial-targeting hybrid vesicles as UA-Mito@HEVs.

[0023] Hybrid extracellular vesicles (HEVs) are created by fusing bone marrow mesenchymal stem cell extracellular vesicles (BVs) and endothelial cell extracellular vesicles (EVs), combining the strengths of both. BVs possess potent tissue repair capabilities and promote mitophagy, thereby improving mitochondrial regeneration and activity in damaged tissues. Furthermore, adhesion molecules and membrane receptors endow EVs with inherent endothelial cell targeting properties, promoting targeted vascular and interendothelial transport. HEVs, through membrane fusion, inherit the potential of BVs to promote mitophagy and regeneration, as well as the vascular targeting capabilities of EVs. This allows for precise targeting of drug-loaded mitochondria to endothelial cells, mitigating the vulnerability of endothelial cells to mitochondrial and endoplasmic reticulum stress under pathological conditions.

[0024] Prior to isolating mitochondria (Mito), bone marrow mesenchymal stem cells (BMSCs) were pretreated with urolithin A, a natural mitochondrial autophagy activator. This pretreatment yielded high-quality mitochondria with excellent structural integrity, respiratory activity, and mitophagy induction capacity. The pretreated mitochondria were then loaded into human endoblasts (HEVs) via extrusion membrane fusion, generating mitochondrial-targeting hybrid vesicles UA-Mito@HEVs with efficient mitochondrial loading and stable structures. These mitochondrial-targeting hybrid vesicles, UA-Mito@HEVs, operate through a dual mechanism: delivering exogenous functional mitochondria to replenish the mitochondrial pool and restore cellular energy supply, while urolithin A pretreatment enhances mitochondrial signal reception, promoting the clearance of damaged intracellular mitochondria and alleviating endoplasmic reticulum stress by restoring redox and calcium balance on mitochondrial-associated membranes (MAMs).

[0025] The therapeutic efficacy of UA-Mito@HEVs was evaluated using two representative disease models characterized by mitochondrial and endoplasmic reticulum dysfunction: diabetic wound healing and acute respiratory distress syndrome (ARDS). In diabetic wounds, chronic hyperglycemia-induced oxidative stress impairs mitochondrial endothelial metabolism and delays angiogenesis; while in ARDS, severe inflammation and oxidative damage lead to mitochondrial collapse, endoplasmic reticulum stress, and vascular exudation. In both cases, UA-Mito@HEVs restored mitochondrial reservoir homeostasis, alleviated endoplasmic reticulum stress, thereby reducing oxidative stress and ultimately promoting tissue repair.

[0026] In summary, the UA-Mito@HEVs provided in this application serve as a highly efficient and biocompatible therapeutic platform that combines targeted delivery to mitochondria with activation of mitophagy. Leveraging the regenerative capabilities of vascular endothelial cells (BVs) and the vascular endothelial cell targeting ability of EVs, UA-Mito@HEVs offer a promising and translational strategy for addressing diseases caused by mitochondrial dysfunction and endoplasmic reticulum stress.

[0027] In some preferred embodiments, the mass ratio of the bone marrow mesenchymal stem cell extracellular vesicles to the endothelial cell extracellular vesicles is (0.5-2):1.

[0028] In some preferred embodiments, the mass ratio of the mixed extracellular vesicles to the loading material is in the range of 1:(2-4).

[0029] In some preferred embodiments, the amount of urolithin A and the bone marrow mesenchymal stem cells treated with it is such that 8 mL of 4-8 μmol / L urolithin A is added to every 2 to 5 million bone marrow mesenchymal stem cells.

[0030] This application also provides a method for preparing mitochondrial-targeted hybrid vesicles, which includes the following steps: 101: Mix bone marrow mesenchymal stem cell extracellular vesicles and endothelial cell extracellular vesicles, and squeeze out the mixed extracellular vesicles.

[0031] In step 101, the conditions for mixing the bone marrow mesenchymal stem cell extravesicles and endothelial cell extravesicles include: performing 4 to 6 cycles of ultrasound treatment in an ice-water bath, with each cycle of ultrasound treatment including an ultrasound amplitude of 20% to 40%, on for 20 to 40 seconds, and off for 1.5 to 2.5 minutes.

[0032] For example, as an example, the conditions for each cycle of ultrasound treatment include an ultrasound amplitude of 30%, on for 30 seconds, and off for 2 minutes.

[0033] Extrusion was performed using a polycarbonate membrane with a pore size of 0.2 μm, and the extrusion was repeated 10 times to obtain mixed extracellular vesicles.

[0034] Mixed extracellular vesicles were incubated at 36.3°C to 37.2°C for 45 to 75 minutes to promote membrane fusion. For example, the mixed extracellular vesicles were incubated at 37°C for 1 hour.

[0035] 102: A loading material comprising mitochondria was isolated from bone marrow mesenchymal stem cells pretreated with urolithin A.

[0036] 103: The loading material is mixed with the mixed extracellular vesicles by extrusion to obtain mitochondrial-targeted hybrid vesicles.

[0037] After mixing by extrusion, the preparation method further includes: incubating at 36.3℃~37.2℃ for 2 hours to obtain a crude product; centrifuging the crude product at 2000~3500×g at 4℃ for 45 minutes to remove large particles; then centrifuging at 4000~10000×g at 4℃ for 45 minutes to separate the vesicles; and then ultracentrifuging the obtained supernatant at 110000~200000×g at 4℃ for 70 minutes to collect nanoscale mitochondrial-targeting hybrid vesicles.

[0038] The nanovesicles were washed twice with sterile PBS, then resuspended in PBS and stored at -80°C for later use.

[0039] The following examples illustrate this.

[0040] For ease of understanding, the sources of the reagents in this application are as follows: Purchase methylglyoxal (MGO), urolithin A (UA), protease inhibitor mixture and glucose from MCE (Shanghai, China).

[0041] Lipopolysaccharide (LPS, E. coli O111:B4 origin) was purchased from Sigma-Aldrich (USA).

[0042] Purchase membrane protein extraction kits, penicillin-streptomycin solution, fetal bovine serum (FBS), Hank's balanced salt solution (HBSS), and Dulbecco modified Eagle's medium (DMEM) from Gibco (USA).

[0043] We sourced DiR, 4′,6-diamide di-benzindole (DAPI), 2′,7′-dichlorofluorescein diacetate (DCFH-DA), Griess kits, BCA detection kits, DiO, DiI, DiD, and the Green-488 actin tracker from Beyotime (China).

[0044] All other chemical reagents and solvents were provided by China National Pharmaceutical Chemical Reagent Co., Ltd.

[0045] Example 1: Cell lines and culture Cultivation of mouse bone marrow mesenchymal stem cells (BMSCs). After aseptic resection of the femur and tibia, long bone marrow samples from 8-week-old C57BL / 6 mice were rinsed with phosphate-buffered saline (PBS) using a sterile syringe. Bone marrow samples were centrifuged at 1000×g for 5 minutes and resuspended in minimum essential α-modified Eagle's medium supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin. The samples were then incubated at 37°C and 5% CO2 for 48 hours. Subsequently, non-adherent cells were discarded, and adherent cells were passaged to approximately 80% (P1).

[0046] Human umbilical vein endothelial cells (HUVECs, #GDC166), HaCaT cells (#GDC106), and 293T cells (#GDC0187) were purchased from the China Center for Type Culture Collection (CCTCC).

[0047] Immortalized mouse bone marrow macrophages (iBMDMs, #M3-1001) were obtained from OriCell in Guangzhou, China.

[0048] Example 2: Animal Experiment Male C57BL / 6 mice aged 8 to 10 weeks were purchased from the Hubei Provincial Center for Disease Control and Prevention (Wuhan, China). All specific pathogen-free (SPF) mice were housed at the Animal Center of Huazhong University of Science and Technology for experiments on diabetic wounds (type 1 diabetes) and ARDS under controlled pathogen-free conditions. Animal experiments were conducted in accordance with the principles approved by the Animal Care and Use Committee (IACUC) of Tongji Medical College, Huazhong University of Science and Technology (Agreement No.: 4452

[2024] ).

[0049] Diabetic wound (type 1 diabetes) model Eight-week-old male C57 / BL6 mice were intraperitoneally injected with streptozotocin (STZ, 50 mg / kg) for five consecutive days. Blood glucose levels were measured using a glucometer at week 1 and week 2 post-injection. Mice with persistent blood glucose levels above 16.7 mM were confirmed as a successfully established diabetic model.

[0050] After successfully establishing and maintaining a diabetic state for 4 weeks, a full-thickness skin wound was created on the back of the mice. Preoperatively, 30 diabetic mice were anesthetized with sodium pentobarbital (Sigma-Aldrich, 1%, 50 mg / kg). After skin preparation and disinfection, a 10 mm diameter full-thickness excision wound was created on the back of each mouse.

[0051] Wounds were treated with PBS, UA-Mito, UA-Mito@EVs, UA-Mito@BVs, or UA-Mito@HEVs, respectively. Each wound received a subcutaneous injection of 50 μg of the corresponding vesicle formulation. Wound healing was continuously monitored and analyzed using ImageJ software.

[0052] UA-Mito refers to the mitochondria extracted from bone marrow mesenchymal stem cells (BMSCs) after pretreatment with urolithin A.

[0053] The difference between the three vesicle formulations UA-Mito@EVs, UA-Mito@BVs and UA-Mito@HEVs lies in the different extracellular vesicles. The three are EVs, BVs and HEVs, which are hybrid extracellular vesicles obtained by fusing EVs and BVs, respectively.

[0054] Example 3: Flow Cytometry Analysis Surface markers of BMSCs were analyzed by flow cytometry. Cells were co-incubated with the following antibodies: CD29, CD34, CD45, and Sca-1 (all purchased from Wuhan Sanying). After antibody binding, samples were collected using a FACSCalibur flow cytometer (BD Biosciences, Franklin Lake, New Jersey, USA), and data were analyzed using FlowJo software (Tree Star Inc., Ashland, Oregon, USA).

[0055] Example 4: Isolation of naked mitochondria According to the manufacturer's instructions, mitochondria were isolated from mouse bone marrow mesenchymal stem cells using a mitochondrial isolation kit (Protein Tech, Wuhan, China, PK10016). It should be noted that the bone marrow mesenchymal stem cells were pretreated with urolithin A, and the dosage of urolithin A and the amount of urolithin A added to the treated bone marrow mesenchymal stem cells was: 8 mL of 6 μmol / L urolithin A was added to every 3 million bone marrow mesenchymal stem cells.

[0056] The specific procedure is as follows: First, for a 10cm culture dish, add 2ml of trypsin to digest until cells detach extensively. Add 2ml of culture medium to neutralize and centrifuge at 1000rpm. Discard the supernatant and resuspend the cells in an appropriate amount of mitochondrial separation reagent. Homogenize 40 times at 4°C using a glass homogenizer. Then centrifuge at 600×g at 4°C for 10 minutes. Collect the supernatant again and centrifuge at 11000×g at 4°C for 10 minutes. The resulting precipitate is the naked mitochondria (UA-Mito). Resuspend it in mitochondrial preservation solution and store at -80°C for later use.

[0057] Example 5: Manufacturing of UA-Mito@HEVs In short, UA-Mito@HEVs are prepared by loading mitochondria isolated from BMSCs into HEVs via liposome extrusion.

[0058] BMSCs were dissociated using rapid trypsin (Gibco, USA), resuspended in PBS, and diluted to 5 × 10⁻⁶. 6 Cells / mL. Cell suspensions were extruded through a series of NuclearPore™ polycarbonate membranes (pore sizes of 10 μm, 5 μm, and 1 μm) at room temperature and under pressure using a small extruder (Avanti Polar Lipids, USA). Each extrusion step was repeated 10 times to obtain bone marrow mesenchymal stem cell extravesicular vesicles (BVs).

[0059] Extracellular vesicles (EVs) derived from HUVECs are also manufactured in a similar manner.

[0060] Preparation of hybrid extracellular vesicles (HEVs): BVs and EVs were first prepared separately by extrusion. Then, the two types of vesicles were mixed at a 1:1 mass ratio and subjected to vortexing and sonication (in an ice-water bath, sonication amplitude 30%; on for 30 seconds, off for 2 minutes, for a total of 4–6 cycles). The mixture was then extruded through a 0.2-micron polycarbonate membrane using an Avanti mini extruder, repeating the above process 10 times, followed by incubation at 37°C for 1 hour to promote membrane fusion. Finally, the isolated mitochondria were mixed with HEVs and then extruded. The mixture was incubated at 37°C for 2 hours to allow the HEVs to load mitochondria. Vesicles were separated by differential centrifugation. The extruded vesicle suspension was first centrifuged at 3000×g at 4°C for 45 minutes to remove large particles, followed by centrifugation at 10000×g at 4°C for 45 minutes to precipitate and separate the microvesicles. The resulting supernatant was then ultracentrifuged at 140000×g at 4°C for 70 minutes to collect nanoscale mitochondrial-targeting hybrid vesicles UA-Mito@HEVs. The nanoscale mitochondrial-targeting hybrid vesicles were washed twice with sterile PBS, resuspended in PBS, and stored at -80°C for later use.

[0061] Example 6: HEV Fusion Verification To confirm the fusion of BVs and EVs, DiI-labeled BVs (denoted as DiI@BVs) and DiO-labeled EVs (denoted as DiO@EVs) were used in hydration to generate DiI and DiO co-labeled HEVs. The colocalization of DiI and DiO fluorescence was confirmed by laser confocal scanning microscopy (CLSM) and Foster resonance energy transfer technique.

[0062] Example 7: Feature Description of UA-Mito@HEVs The structure of extracellular vesicles was observed using transmission electron microscopy (TEM). A 10 μL suspension of extracellular vesicles was placed on a copper grid and soaked for 5 to 10 minutes; excess liquid was aspirated with filter paper. After air drying, the sample was stained with 2% uranyl acetate (10 μL) and imaged using an HT7700 TEM (Hitachi, Japan). Particle size tracer analysis (NTA) was performed using a Zeta View PMX-120 system (Particle Metrix, Germany) to measure the size and concentration of nanovesicles.

[0063] Example 8: In vivo targeted validation of endothelial cells DiI-labeled HEVs derived from HUVECs (denoted as EC+HEVs, 100 μL, 50 μg / mL) and DiI-labeled HEVs derived from 293T (denoted as 293T, 100 μL, 50 μg / mL) were injected into the wounds of EC+HEVs and 293T+HEVs in diabetic mice, respectively. Wound tissue collected on day 3 was frozen and sectioned longitudinally. These sections were subsequently stained with DAPI and anti-CD31 antibody. Fluorescence microscopy images (Nikon, Japan) were taken at the center and edges of the wounds.

[0064] Example 9: Cell Uptake Validation DiI-labeled vesicles were incubated with HUVECs for 24 hours. Cells were stained with phalloidin and DAPI to observe the cytoskeleton and nucleus. Images were taken using a Nikon confocal microscope. Co-localization of DiI and DiO in fusion vesicles was analyzed using ImageJ.

[0065] Example 10: In vitro experimental analysis of inflammation-targeting effects Endothelial cells were seeded in 24-well plates and cultured for 24 hours, followed by treatment with TNF-α (20 ng / mL) or PBS for 12 hours. Cells or extracellular vesicles were then pretreated according to specific experimental requirements. Endothelial cells from different groups were pretreated with anti-ICAM1 antibody (Wuhan Sanying) or control IgG (Wuhan Sanying) for 2 hours, followed by treatment with different extracellular vesicles for 12 hours. Cells were then imaged using confocal microscopy or collected for flow cytometry analysis.

[0066] To investigate the role of ITGB2 (Wuhan Sanying), extracellular vesicles were first treated with anti-ITGB2 antibody or pretreated with IgG as a control, and then co-incubated with endothelial cells. Unbound antibodies were removed by ultracentrifugation, and then different vesicles were added according to the group and co-cultured with endothelial cells for 12 hours before subsequent analysis.

[0067] Example 11: Cell proliferation and migration Cell proliferation was detected using the EdU assay kit, while cell migration was assessed using Transwell assays and scratch assays. HUVECs were treated with 400 μmol MGO to simulate oxidative stress. CCK-8 and calcein AM / PI double staining was performed. Images of stained cells were captured using a fluorescence microscope.

[0068] Example 12: Measurement of Oxidative Stress ROS levels were measured using DCFH-DA. MDA levels, as well as the activities of SOD and GPx, were analyzed using the corresponding assay kits.

[0069] Example 13: Apoptosis assay HUVEC apoptosis was analyzed using the Annexin V-FITC apoptosis detection kit. Stained cells were evaluated by flow cytometry and analyzed using FlowJo software.

[0070] Example 14: mRNA sequencing analysis process The library was sequenced on an Illumina Novaseq 6000 sequencing platform, generating 150 bp paired-end reads. First, the raw reads in FastQ format were processed using FastP software to remove low-quality reads and obtain high-quality clean reads. The high-quality reads were then aligned to a reference genome using HISAT2 software, and read counts for each gene were calculated using HTSeq-count software. Gene expression levels were normalized using FPKM (number of fragments per kilobase transcript per million reads).

[0071] Principal component analysis (PCA) was performed using R software (v 3.2.0) to assess the biological repeatability of the samples. Differential expression analysis was performed using DESeq2 software (5), with Q value < 0.05 and fold change > 2 or < 0.5 set as the screening threshold for significantly differentially expressed genes (DEGs). Hierarchical cluster analysis was performed on differentially expressed genes using R software (v 3.2.0) to show the gene expression patterns among different groups and samples; radar charts of the top 30 differentially expressed genes were plotted using the ggradar package in R language to visually present the expression characteristics of upregulated and downregulated differentially expressed genes.

[0072] Based on hypergeometric distribution, R software (v 3.2.0) was used to perform Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Reactome, and WikiPathways pathway enrichment analyses on differentially expressed genes. Significantly enriched functional entries were screened, and bar charts, chord charts, and bubble charts of significantly enriched entries were generated using R software (v 3.2.0).

[0073] Gene set enrichment analysis (GSEA) was performed using GSEA software. This analysis used a predefined gene set, ranked genes according to their differential expression levels in the two types of samples, and then examined whether the predefined gene set was significantly enriched at the top (high expression end) or bottom (low expression end) of the ranked list.

[0074] Example 15: Quantification of Mitochondrial DNA Mitochondrial DNA (mtDNA) analysis was performed using a commercial kit (BLT, MTD002). Mitochondrial DNA was extracted from cells / tissues using the kit and then quantified according to the kit instructions.

[0075] Example 16: Mitochondrial morphology assessment Mitochondrial morphology was assessed using mCherry-labeled HUVECs stable cell lines. Cells were directly imaged using confocal imaging following specified treatments, including MGO-induced damage and various therapeutic interventions. Mitochondrial structure and network were observed using a confocal laser scanning microscope (LSM900, Zeiss, Germany). Mitochondrial morphology parameters were quantitatively analyzed using ImageJ software with the MINA 2.0 plugin.

[0076] Example 17: Endoplasmic Reticulum-Mitochondrial Contact Analysis Cells stably expressing ER-DsRed were selected for endoplasmic reticulum observation, and mitochondria were labeled in stably expressing mitochondrial green-stained cell lines. Confocal images were acquired for each group under identical settings using a Carl Zeiss LSM900 confocal microscope. ER-mitochondrial colocalization was quantitatively analyzed using the Pearson correlation coefficient, a widely accepted indicator for assessing signal overlap between two fluorescence channels. The Pearson coefficient was calculated automatically using ZEN software (Carl Zeiss).

[0077] Example 18: Thioflavin T (ThT) staining Thioflavin T (ThT) staining was performed to assess endoplasmic reticulum (ER) stress-related protein aggregation in HUVECs. HUVECs were seeded on glass coverslips and placed in 24-well plates, subsequently subjected to MGO-induced damage and / or related treatments. After culture, cells were washed twice with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde at room temperature for 15–20 min. After three washes with PBS, cells were incubated with ThT working solution (1:1000 dilution in PBS) at room temperature in the dark for 15 min. Excess dye was removed by washing three times with PBS. Nuclei were stained with DAPI for 5–10 min. Fluorescence images were captured using confocal fluorescence microscopy and measured according to the ThT excitation / emission settings. ThT fluorescence intensity reflected the degree of protein aggregation and was quantified using ImageJ software. Total protein from SDS-PAGE and Western blotting of vesicles was extracted using RIPA dissolution buffer supplemented with protease inhibitors (Roche, Switzerland). Protein concentration was determined using a BCA protein assay kit (Beyotime, Shanghai, China). Equal amounts of total protein (20 μg) were separated by SDS-PAGE (Beyotime, China). Proteins in the polyacrylamide gel were detected using Coomassie blue staining (Hycezmbio, China).

[0078] For the Western blotting method, total protein was separated by SDS-PAGE and transferred to a polyvinyl chloride membrane (Millipore, USA). The membrane was then sealed and incubated overnight at 4°C with the corresponding primary antibody. Subsequently, these membranes were incubated with secondary antibody for 2 hours and exposed to X-rays (UVP, CA, USA).

[0079] Example 19: Acute Respiratory Distress Syndrome Model All animal experiments have been approved by the Animal Care Committee of Tongji Medical College.

[0080] Mice were anesthetized with xylthiazole / ketamine and induced with lipopolysaccharide (LPS) (2 mg / kg body weight) via intratracheal infusion. Four hours post-injury, PBS, UA-Mito, UA-Mito@EVs, UA-Mito@BVs, or UA-Mito@HEVs were administered via intratracheal infusion. Twenty-four hours post-injury, mice were euthanized, and lung tissue was harvested for analysis or preparation of Precision Cut Lung Slices (PCLS).

[0081] Example 20: Histological and Immunofluorescence Analysis Wound tissue was fixed and sectioned by embedding in paraffin. Granulation tissue and collagen deposition were assessed by hematoxylin and eosin (HE) and Massen staining, respectively. Immunofluorescence staining was performed for CD31, ATF4, and P62, and the data were analyzed using ImageJ.

[0082] After lung tissue fixation, the degree of lung injury was assessed using hematoxylin and eosin (HE). Immunofluorescence staining was performed for CD31, ATF4, and P62, and the data were analyzed using ImageJ.

[0083] Statistical data Statistical analysis was conducted using GraphPad Prism software (version 8.4.3, La Jolla, California, USA). Independent samples t-tests were used for comparisons between two groups; one-way ANOVA combined with Tukey's post-hoc test was used for comparisons between three or more groups. The statistical significance threshold was set at p < 0.05. Data are presented as mean ± standard deviation (mean ± SDs). * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and ns indicates no significant difference.

[0084] In summary, this study has developed a bioengineered mitochondrial therapeutic, UA-Mito@HEVs, capable of restoring mitochondrial pool homeostasis in various injury models. UA-Mito@HEVs delivers both normal mitochondria and urolithin A with mitochondrial activation function via vesicles, overcoming key limitations of traditional mitochondrial transplantation, including short cell retention time and functional impairment. This comprehensive strategy simultaneously replenishes functional mitochondria and effectively removes damaged organelles, thereby stabilizing the mitochondrial network and maintaining cellular energy under pathological conditions. Through this coordinated regulation, UA-Mito@HEVs effectively mitigates oxidative damage and promotes the recovery of endothelial and tissue function. In diabetic wound healing and acute lung injury models, the restoration of mitochondrial pool balance by UA-Mito@HEVs represents a robust and universally applicable therapeutic principle. Overall, this work provides a new concept and technological framework for organelle-based nanotherapy, where drugs combining mitochondrial delivery and post-mitochondrial uptake activation can be used to treat inflammatory and degenerative diseases caused by mitochondrial dysfunction.

[0085] Figure 1 The preparation and characterization results of UA-Mito@HEVs are as follows: (A) is a transmission electron microscope (TEM) image, scale bar: 200 nm; (B) shows the particle size distribution of vesicles in phosphate-buffered saline (PBS); (C) represents the zeta potential of vesicles in PBS; (D) Verification of HEV fusion using fluorescence resonance energy transfer (FRET) effect; (E) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of extracellular endothelial cells (EVs), bone marrow mesenchymal stem cell extracellular vesicles (BVs) and HEVs. (F) is a laser confocal scanning microscopy (CLSM) image of fluorescently labeled HEVs (red: DiI-labeled BVs; green: DiO-labeled EVs), scale bar: 5 μm; (G) is a representative confocal microscopy image of UA-Mito@HEVs (HEV membranes are labeled with DiI and appear red; mitochondria within HEVs are stained with mitochondrial green fluorescent protein (Mito-GFP) and appear green), scale bar: 20 micrometers; (H) Detection of exogenous mitochondrial delivery in a live-cell system (recipient cells: mitochondrial-cherry-red fluorescent protein (Mito-Cherry) labeled human umbilical vein endothelial cells (HUVECs); donor cells: Mito-GFP labeled bone marrow mesenchymal stem cells (BMSCs)), scale bar: 20 micrometers; (I) is sodium-potassium ATPase (Na) + -K + Western blot analysis of proteins containing ATPase and cytochrome c oxidase subunit IV (COX IV) (n=3) (J) shows the flow cytometry (FCM) analysis of Mito-GFP in heterologous mitochondria (Iso-Mito), UA-Mito, or UA-Mito@HEVs.

[0086] Figure 2 The results of UA-Mito@HEVs' internalization and targeted uptake are shown, including: (A) represents grouping information; (B) is a fluorescence image of human umbilical vein endothelial cells (HUVECs) uptake of HEVs from 293T cells (293T+HEVs), HEVs from HUVECs (EC+HEVs), HEVs from HUVECs pretreated with anti-CXCR4 (EC+HEVs+Anti-CXCR4), and HEVs from HUVECs pretreated with immunoglobulin G (IgG) (EC+HEVs+IgG). Scale bar: 100 micrometers; (C) Quantitative results of HUVECs cell uptake; (D) is a representative histogram and quantitative analysis (n=3); (E) Validation of vesicle-targeted delivery in an in vitro skin cell model of human dermal fibroblasts (HDF), scale bar: 100 micrometers; (F) Validation of vesicle-targeted delivery in an in vitro skin cell model of human immortalized keratinocytes (Hacat), scale bar: 100 micrometers; (G) represents flow cytometry (FCM) analysis (n=3).

[0087] Figure 3 The in vitro efficacy results of UA-Mito@HEVs in promoting cell proliferation, migration, and survival under oxidative microenvironment include: (A) A representative image of proliferating HUVECs detected by the grouping information and the 5-ethynyl-2'-deoxyuridine (EdU) staining method; (B) Statistical analysis of EdU fluorescent positive cells (n=6), scale bar: 100 micrometers; (C) is a representative image of HUVECs proliferating in the Transwell experiment; (D) is the statistical analysis of HUVECs proliferation in the Transwell experiment (n=3); (E) Scratch test results of HUVECs at 0 hours and 24 hours after different vesicle treatments; (F) is the corresponding statistical analysis of wound healing rate (n=6), scale bar: 100 micrometers; (G) is a representative image from the HUVECs tube formation experiment; (H) is a statistical analysis of the overall tube length in the HUVECs tube formation experiment (n=5), scale bar: 100 micrometers; (I) Analysis of HUVECs by 2',7'-dichlorofluorescein diacetate (DCFH-DA) staining; (J) shows the corresponding statistical analysis and flow cytometry (FCM) detection of relative DCFH fluorescence intensity (n=6), scale bar: 100 micrometers; (K) represents the flow cytometry analysis of DCFH-DA in each group (n=6). (L) represents the malondialdehyde (MDA) activity level in endothelial cells after different treatments (n=6); (M) represents the superoxide dismutase (SOD) activity level in endothelial cells after different treatments (n=6).

[0088] Figure 4 The transcriptomic results of HUVECs treated with UA-Mito@HEVs under oxidative stress are presented in the principal component analysis. The principal component analysis (PCA) showed that there were significant differences between the UA-Mito@HEVs group and the control group.

[0089] Figure 5 This is a transcriptomic analysis of HUVECs treated with UA-Mito@HEVs under oxidative stress, focusing on the processing of endoplasmic reticulum stress-related proteins in the gene set enrichment analysis (GSEA). The analysis showed that the processing of endoplasmic reticulum stress-related proteins was downregulated in the gene set enrichment analysis (GSEA).

[0090] Figure 6 Transcriptomic analysis of HUVECs treated with UA-Mito@HEVs under oxidative stress revealed positive enrichment of mitophagy in the gene set enrichment analysis (GSEA).

[0091] Figure 7 The transcriptomic analysis results of HUVECs treated with UA-Mito@HEVs under oxidative stress on autophagy in animal cells in GSEA show that autophagy in animal cells was positively enriched in gene set enrichment analysis (GSEA).

[0092] Figure 8 The transcriptomic analysis results of HUVECs treated with UA-Mito@HEVs under oxidative stress regarding autophagosome formation in the gene set enrichment analysis (GSEA) show positive enrichment of autophagosome formation.

[0093] Figure 9 Transcriptomic analysis of HUVECs treated with UA-Mito@HEVs under oxidative stress revealed the following results regarding mitochondrial respiratory chain complex I in the gene set enrichment analysis (GSEA).

[0094] Figure 10 The results of UA-Mito@HEVs improving mitochondrial bioenergetics under methylglyoxal (MGO) stress include: (A) Analysis of mitochondrial DNA (mtDNA) staining in HUVECs; (B) Statistical analysis of the relative fluorescence intensity of mtDNA (n=5), scale bar: 100 micrometers; (C) is a representative image of JC-1 fluorescent staining; (D) Flow cytometry analysis of JC-1 in each group (n=3), scale bar: 20 micrometers; (E) Quantitative analysis of the green / red fluorescence ratio of JC-1 (n=3); (F) represents the content of adenosine triphosphate (ATP); (G) is a representative image of mitochondrial superoxide probe (MitoSOX) fluorescent staining; (H) shows the flow cytometry analysis of MitoSOX in each group (n=3), scale bar: 20 micrometers; (I) Quantitative analysis of MitoSOX (n=3).

[0095] Figure 11 The results show the regulation of mitophagy and endoplasmic reticulum-mitochondria by UA-Mito@HEVs, including: (A) A representative fluorescence image of the mitochondrial network in a stable mitochondrial cell line; (B) For quantitative analysis (n=3), scale bar: 20 micrometers; (C) is a representative fluorescence image of co-localization of mitochondria and endoplasmic reticulum; (D) Quantitative analysis of Pearson correlation coefficient (n=3), scale bar: 20 micrometers; (E) represents mitochondrial calcium ions (Ca). 2+ )content; (F) HUVECs were analyzed by thioflavin T (THT) staining; (G) is the corresponding statistical analysis of the relative fluorescence intensity of THT (n=3), scale bar: 100 micrometers; (H) Western blot analysis of P62, Parkin, microtubule-associated protein 1 light chain 3 (LC3I / II), and PTEN-induced kinase 1 (PINK) in HUVECs after different treatments (n=3). (I) Western blot analysis of protein kinase R-like endoplasmic reticulum kinase (PERK), phosphorylated PERK (p-PERK), activated transcription factor 4 (ATF4) and C / EBP homolog (CHOP) in HUVECs after different treatments (n=3).

[0096] Figure 12 UA-Mito@HEVs significantly promoted the healing outcomes of diabetic wounds, including: (A) shows the grouping information and representative images; (B) For wound healing monitoring; (C) Bar chart showing wound closure rates at 0, 3, 7, and 14 days. (D) is a line graph showing the wound closure rate at 0, 3, 7, and 14 days. (E) Heat maps of wound closure rates at 0, 3, 7 and 14 days (n=6); (F) is a representative image of hematoxylin-eosin (HE) staining of wound tissue at 12 days; (G) represents the corresponding statistical analysis of collagen deposition in wound tissue (n=3); (H) is a representative image of Masson staining in wound tissue at 12 days, scale bar: 20 micrometers; (I) is the corresponding statistical analysis of wound healing width in wound tissue (n=3).

[0097] Figure 13 Histological and immunofluorescence analysis results for diabetic wound healing, including: (A) is a grouping information and a representative image of CD31 immunofluorescence staining in wound tissue (n=3), scale bar: 20 micrometers; (B) is the corresponding statistical analysis of relative fluorescence intensity; (C) Dihydroethidium (DHE) staining of wound sections from each group 14 days after treatment (to assess the level of reactive oxygen species (ROS) in wound tissue) (n=3), scale bar: 20 micrometers; (D) Statistical analysis of dihydroethidium (DHE) staining of wound sections in each group 14 days after treatment; (E) is a representative image of P62 immunofluorescence staining (n=3), scale bar: 20 micrometers; (F) represents the statistical analysis of immunofluorescence staining of P62; (G) is a representative image of immunofluorescence staining of ATF4 and dynamism-related protein 1 (DRP1) (n=3), scale bar: 20 micrometers; (H) and (I) are statistical analyses of immunofluorescence staining of ATF4 and DRP1.

[0098] Figure 14 UA-Mito@HEVs significantly reduced lung injury outcomes in acute respiratory distress syndrome (ARDS), including: (A) Macrophage phenotypic polarization study: Flow cytometry (FCM) was used to detect the polarization of inflammatory bone marrow-derived macrophages (iBMDMs) after different vesicle treatments (n=3). (B) Quantitative analysis of the M2 / M1 macrophage ratio after treatment; (C) represents CD86-positive cells (indicating M1 subtype macrophages); (D) are CD206 positive cells (representing M2 subtype macrophages); (E) Images of lung injury after different treatment groups; (F) shows the grouping information and histological images of lung tissue stained with hematoxylin and eosin (H&E). Scale bar: 20 micrometers. (G) represents the total lung injury score (n=3) based on histological images and five pathophysiological features. (H) is a representative image of P62 immunofluorescence staining (n=3), scale bar: 100 micrometers; (I) Statistical analysis of immunofluorescence staining of P62; (J) is a representative image of ATF4 immunofluorescence staining (n=3), scale bar: 100 micrometers; (K) represents the statistical analysis of ATF4 immunofluorescence staining.

[0099] The above description is merely a specific embodiment of this application, enabling those skilled in the art to understand or implement this application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of this application. Therefore, this application is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A hybrid vesicle targeting mitochondria, characterized in that, It includes a mixture of extracellular vesicles and a loading material contained in the mixture of extracellular vesicles, the loading material including mitochondria extracted from bone marrow mesenchymal stem cells pretreated with urolithin A, the mixture of extracellular vesicles including bone marrow mesenchymal stem cell extracellular vesicles and endothelial cell extracellular vesicles; The mass ratio of extracellular vesicles of bone marrow mesenchymal stem cells to extracellular vesicles of endothelial cells is (0.5–2):1; The mass ratio of the mixed extracellular vesicles to the loading material ranges from 1:(2 to 4). The amount of urolithin A added to the bone marrow mesenchymal stem cells treated with it is: 8 mL of 4-8 μmol / L urolithin A added to every 200-500 million bone marrow mesenchymal stem cells. The method for preparing the mitochondrial-targeted hybrid vesicles includes: The extracellular vesicles of bone marrow mesenchymal stem cells and extracellular vesicles of endothelial cells were mixed and the mixed extracellular vesicles were extruded. Loading material was isolated from bone marrow mesenchymal stem cells pretreated with urolithin A; The loading material was mixed with hybrid extracellular vesicles by extrusion to obtain mitochondrial-targeting hybrid vesicles; The conditions for mixing the extracellular vesicles of bone marrow mesenchymal stem cells and extracellular vesicles of endothelial cells include: performing 4 to 6 cycles of ultrasound treatment in an ice-water bath, with each cycle of ultrasound treatment including an ultrasound amplitude of 20% to 40%, on for 20 to 40 seconds, and off for 1.5 to 2.5 minutes. A polycarbonate membrane with a pore size of 0.2 μm was extruded, and the extrusion was repeated 10 times to obtain mixed extracellular vesicles; Before mixing the loading material with the mixed extracellular vesicles, the preparation method further includes: incubating the mixed extracellular vesicles at 36.3℃~37.2℃ for 45min~75min to promote membrane fusion; After mixing by extrusion, the preparation method further includes: incubating at 36.3℃~37.2℃ for 2 hours to obtain crude product; centrifuging the crude product at 2000~3500×g at 4℃ for 45 minutes, followed by centrifugation at 4000~10000×g at 4℃ for 45 minutes; and then ultracentrifuging the obtained supernatant at 110000~200000×g at 4℃ for 70 minutes to collect nanoscale mitochondrial-targeting hybrid vesicles.

2. The use of a mitochondrial-targeting hybrid vesicle as described in claim 1 in the preparation of a medicament for treating diabetic wounds or acute respiratory distress syndrome.

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