Mitochondria-enriched extracellular vesicles, and preparation method and application thereof

Abstract

This invention relates to mitochondrial-enriched extracellular vesicles, their preparation methods, and applications, which can effectively solve the drug problem in spontaneous intracerebral hemorrhage. The technical solution involves using platelets as raw material, and obtaining platelet-derived extracellular vesicles carrying active mitochondria through activation induction, differential centrifugation and fractionation, membrane filtration and elution enrichment. This achieves efficient and stable loading of functional mitochondria, and the resulting PEV Mito has a complete structure and uniform particle size, which can effectively maintain mitochondrial membrane potential and biological activity. It can achieve efficient delivery and intracellular internalization to damaged neurons and restore mitochondrial function. The PEV Mito prepared by this invention has high delivery efficiency and excellent biocompatibility, and can be used to prepare drugs for the treatment of intracerebral hemorrhage and secondary neurological injury, with good prospects for clinical translation.

Description

Technical Field

[0001] This invention relates to the field of mitochondrial delivery carrier preparation technology, and in particular to a method for preparing and applying mitochondrial-enriched extracellular vesicles. Background Technology

[0002] Spontaneous intracerebral hemorrhage (ICH) is an extremely serious type of stroke, causing significant disability and mortality worldwide. ICH accounts for approximately 10% to 15% of all stroke cases, with a 30-day mortality rate exceeding 40%, and importantly, most survivors suffer from long-term disability. Current clinical treatment strategies for ICH include surgical hematoma removal and supportive care aimed at slowing hematoma growth and controlling intracranial pressure. While these interventions can mitigate the acute mass effect, they are not effective in addressing the widespread neuronal damage and dysfunction following hemorrhagic brain injury. Neuroprotective pharmacological treatments, such as N-methyl-D-aspartate (NMDA) receptor antagonists or free radical scavengers, have shown limited effectiveness in clinical trials and cannot reverse existing neuronal loss. New research evidence suggests that neurons in the peri-hemorrhagic region of the brain are characterized by widespread mitochondrial dysfunction. Disruption of the mitochondrial network structure and impaired bioenergy metabolism lead to neuronal death and neurological dysfunction. However, current strategies primarily focus on modulating downstream stress responses after injury, failing to directly restore the structural and functional integrity of damaged mitochondria.

[0003] Studies of central nervous system injury models have revealed that glial cells can achieve endogenous rescue by transferring mitochondria to damaged neurons, highlighting the importance of organelle-level intervention. Mitochondrial transplantation has demonstrated tissue protection and repair potential in various preclinical models and has been explored in early clinical trials in areas such as myocardial ischemia-reperfusion injury, suggesting the feasibility of exogenous mitochondrial supplementation in improving cellular energy metabolism and tissue function. It is considered a potential therapeutic strategy for restoring neuronal energy homeostasis and reducing secondary neurological damage after cerebral hemorrhage. However, free mitochondria are susceptible to mechanical stress, oxidative environments, and immune clearance during isolation, preservation, and in vivo delivery, leading to decreased stability, difficulty in maintaining function, and limited cellular uptake efficiency. These issues restrict their potential for clinical translation. Developing carriers with natural delivery capabilities and better biocompatibility has become an important research direction in this field.

[0004] Platelets are anucleated blood cells with a diameter of approximately 2-3 μm. Their cytoplasm is rich in functional mitochondria, which rely on oxidative phosphorylation to maintain energy metabolism and physiological functions. This invention constructs an extracellular vesicle (PEV-Mito) derived from platelets and rich in mitochondrial components as an in vivo mitochondrial delivery system for replenishing mitochondrial function in neurons damaged after cerebral hemorrhage. Compared with free mitochondria, PEV-Mito utilizes natural membrane vesicles as a protective and delivery carrier, significantly improving the stability and delivery feasibility of mitochondrial-related components in the in vivo environment, providing a safe and efficient new mitochondrial replenishment therapy for secondary brain injury after cerebral hemorrhage. Summary of the Invention

[0005] Based on the above technical background, the first objective of this invention is to provide a mitochondrial-enriched extracellular vesicle; the second objective of this invention is to provide a method for preparing mitochondrial-enriched extracellular vesicles; and the third objective of this invention is to provide the application of mitochondrial-enriched extracellular vesicles in the preparation of treatments for cerebral hemorrhage, improvement of neuronal mitochondrial dysfunction, and mitochondrial transplantation; which can effectively solve the problem of medication for spontaneous intracerebral hemorrhage.

[0006] The first objective of this invention is to provide a mitochondrial-enriched extracellular vesicle, wherein the vesicle is derived from extracellular vesicles formed by the in vitro activation and release of platelets, and the vesicle is enriched with mitochondria or mitochondrial-related components that have complete structure and functional activity.

[0007] Preferably, the platelets are derived from the blood of mammals, including but not limited to the blood of rats, humans, rabbits, and pigs.

[0008] Preferably, the mitochondria in the vesicles have intact membrane potential, the red / green fluorescence ratio of the mitochondrial membrane potential fluorescent probe (JC-1) is higher than that of ordinary extracellular vesicles (PEVs), and the content of adenosine triphosphate (ATP) is higher.

[0009] A second objective of this invention is a method for preparing mitochondrial-enriched extracellular vesicles, comprising the following steps: 1) Collect whole blood from mammals and treat it with an anticoagulant to obtain anticoagulated whole blood; 2) Centrifuge the anticoagulated whole blood from step 1) and collect the supernatant platelet-rich plasma (PRP). 3) Add stabilizing buffer to the PRP in step 2) and mix well to inhibit non-specific platelet activation; 4) Centrifuge the PRP processed in step 3) and wash it with washing buffer to obtain purified platelet precipitate; 5) Activate the platelets from step 4) to release vesicles containing mitochondria or mitochondrial-related components; 6) The activated system from step 5) was centrifuged and membrane filtered to separate crude PEV and crude PEV-Mito, respectively. 7) The crude vesicles from step 6) were purified by ultracentrifugation to obtain the target vesicle product.

[0010] Preferably, in step 1), the anticoagulant is sodium citrate, preferably a sodium citrate solution with a mass fraction of 3%-4%.

[0011] More preferably, in step 1), the volume ratio of anticoagulant to whole blood is 1:8 to 1:10, and anticoagulation is achieved by gently inverting and mixing after blood collection.

[0012] Preferably, in step 2), the centrifugation temperature is 0-10 ℃, the centrifugation speed is 1000-2000 rpm, the centrifugation time is 10-15 min, and the upper plasma layer is collected as platelet-rich plasma.

[0013] Preferably, in step 3), the stabilizing buffer contains ethylenediaminetetraacetic acid (EDTA) and prostaglandin E1 (PGE1) at concentrations of 0.5-2 mM and 1-5 μM, respectively, and the volume ratio of the stabilizing buffer to PRP is 1:3–1:10.

[0014] Preferably, in step 4), the washing buffer contains EDTA and phenyl methylsulfonyl fluoride (PMSF) at concentrations of 0.5-2 mM and 1-5 mM, respectively.

[0015] More preferably, in step 4), the centrifugation conditions are 3000-5000 rpm for 15-25 min, the washing is performed 2-4 times, and the sample is resuspended in sterile phosphate buffer (PBS).

[0016] Preferably, in step 5), the activation agent is thrombin, the concentration of thrombin is 0.5-2 U / mL, the activation temperature is 35-40 ℃, and the activation time is 30-90 min.

[0017] Preferably, in step 6), centrifugation is performed first to remove unactivated platelets and cell debris, with centrifugation conditions of 3000-5000 rpm for 15-25 min.

[0018] More preferably, in step 6), a sterile filter membrane with a pore size of 0.20-0.40 μm is used for separation, the filtrate is crude PEV, and the portion retained by the filter membrane is recovered as PEV-Mito by backwashing with sterile PBS.

[0019] Preferably, in step 7), the crude PEV-Mito product is centrifuged at 10000-30000 ×g and 0-10 ℃ for 30-90 min.

[0020] More preferably, in step 7), after centrifugation, the supernatant is discarded and the sample is resuspended in sterile PBS, and the resulting sample is stored at 0-10 °C.

[0021] A third objective of this invention is the application of the mitochondrial-enriched extracellular vesicles in the preparation of drugs to improve mitochondrial dysfunction-related diseases.

[0022] The diseases mentioned include cerebral hemorrhage, brain injury, ischemia-reperfusion injury, neurodegenerative diseases, and diseases related to mitochondrial defects and deficiencies; the mitochondrial-enriched extracellular vesicles can be taken up by various types of cells, including but not limited to neurons, immune cells, and stem cells.

[0023] The beneficial effects of this invention are as follows: 1. This invention establishes a method for extracting and preparing platelet-derived mitochondrial enriched vesicles. It utilizes natural vesicles released by platelet activation to encapsulate functional mitochondria, eliminating the need for artificially synthesized carriers. This method exhibits good biocompatibility and high safety, avoiding the problems of poor stability and easy inactivation associated with delivery of free mitochondria.

[0024] 2. The platelet-derived mitochondrial enrichment vesicles of the present invention can be efficiently taken up by damaged neurons and co-localized with endogenous mitochondria, directly replenishing functional mitochondria, restoring mitochondrial membrane potential, increasing cellular oxygen consumption rate (OCR), reducing reactive oxygen species (ROS) accumulation, and effectively alleviating hemin-induced oxidative stress and mitochondrial dysfunction in mouse hippocampal neuronal cell line (HT22).

[0025] 3. The PEV-Mito of the present invention can significantly improve neurological deficits and reduce pathological damage to brain tissue in animal models of cerebral hemorrhage. It can be used to treat various mitochondrial dysfunction and mitochondrial deficiency-related diseases such as cerebral hemorrhage, brain injury, and ischemia-reperfusion injury. It is a safe and effective mitochondrial delivery therapy with good prospects for clinical translation. Attached Figure Description

[0026] Figure 1 These are representative transmission electron microscopy images of PEV-Mito and PEV in this invention. Dense mitochondrial structures are visible inside some vesicles in the PEV-Mito group, while the PEV group mainly exhibits a vesicle-like morphology with a relatively simple internal structure.

[0027] Figure 2 In this invention, the JC-1 fluorescent probe was used to detect the mitochondrial membrane potential in PEV and PEV-Mito samples.

[0028] Figure 3 This diagram illustrates the spatial correlation characteristics within HT22 cells of endogenous mitochondria and exogenous mitochondrial-related signals after PEV-Mito treatment according to the present invention. Red indicates mitochondrial-related signals derived from PEV-Mito, and cyan indicates endogenous mitochondrial signals in HT22 cells. The left image shows the overall view, while the middle and right images are magnified views of specific areas.

[0029] Figure 4 The curves showing the changes in oxygen consumption rate of HT22 cells in each group of this invention are shown.

[0030] Figure 5 This invention describes the changes in reactive oxygen species (ROS) levels in HT22 cells under different treatment conditions.

[0031] Figure 6 To assess the changes in modified neurological severity score (mNSS) scores after cerebral hemorrhage in mice under different treatment groups according to this invention, a modified evaluation method was used to assess the changes in neurological function at different time points after surgery in each group of mice.

[0032] Figure 7 In this invention, a represents the results of the forelimb placement experiment after cerebral hemorrhage in mice of different treatment groups, and b represents the results of the angle turning experiment after cerebral hemorrhage in mice of different treatment groups. The detection time points are 0, 1, 3, 5, 7, 14 and 30 days after surgery. Detailed Implementation

[0033] To clearly present the technical principles, implementation process, and practical effects of this invention, the technical solution of this invention will be described in detail and completely below with reference to the accompanying drawings and specific embodiments. The described embodiments are only some embodiments of this invention, not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the protection scope of this invention. Unless otherwise specified, the materials, reagents, etc., used in the following embodiments are commercially available.

[0034] Example 1: Preparation and structural characterization of platelet-derived mitochondrial enriched vesicles This embodiment provides a method for preparing PEV-Mito and verifies its structure using transmission electron microscopy (TEM), while setting ordinary platelet extracellular vesicles as a control.

[0035] (1) Preparation steps a) Healthy C57BL / 6J mice were anesthetized and whole blood was collected through the orbital venous plexus. 3.8% sodium citrate anticoagulant was pre-placed in the blood collection vessel, and anticoagulation was performed at a ratio of anticoagulant to whole blood volume of 1:9. After blood collection, the blood was gently inverted and mixed, and centrifuged at 2-8 ℃ and 1500 rpm for 12 min. The upper layer of platelet-rich plasma was collected.

[0036] b) Then add a stabilization buffer containing 1 mM EDTA and 2 μM PGE1 to the PRP and gently mix at a ratio of 1 mL PRP to 200 μL of stabilization buffer to reduce non-specific activation of platelets during processing.

[0037] c) After gently mixing the PRP, centrifuge at 4000 rpm for 20 min, discard the supernatant and collect the platelet pellet. Then, gently wash the platelet pellet with a buffer containing 1 mM EDTA and 2 mM PMSF for a total of 3 washes to reduce interference from residual plasma proteins and other soluble impurities.

[0038] d) After washing, platelets were resuspended in PBS to 1 mL, thrombin was added to a final concentration of 1 U / mL, and the platelets were incubated at 37 °C for 60 min to induce platelet activation and release of extracellular vesicles.

[0039] e) After stimulation, the reaction system was centrifuged at 4000 rpm for 20 min to remove unactivated platelets and larger cell debris, and the supernatant was collected for subsequent separation. Considering that the vesicle population released after thrombin stimulation may contain both vesicles derived from ordinary platelets and larger vesicles rich in mitochondrial components, based on the possible particle size differences between the two, a 0.2 μm sterile filter membrane was used for preliminary separation of the supernatant.

[0040] f) The filtrate collected after filtration was defined as the PEV group; the portion retained by the filter membrane was eluted with sterile PBS and recovered as the vesicle PEV-Mito group rich in mitochondrial components. The obtained PEV-Mito samples were centrifuged at 18000 × g for 60 min, the supernatant was discarded and the samples were gently resuspended in sterile PBS.

[0041] (2) Transmission electron microscopy structure verification To observe the morphological and ultrastructural characteristics of platelet-derived vesicles, freshly isolated platelet samples, as well as PEV and PEV-Mito samples obtained through separation and enrichment, were fixed in 2.5% glutaraldehyde fixative at 4 °C for 24 h. After fixation, the samples were washed with PBS, followed by gradient ethanol dehydration, epoxy resin infiltration and embedding, and polymerization curing. The embedded samples were cut into ultrathin sections, stained with uranium acetate and lead citrate, and observed and images were acquired under a transmission electron microscope for comparison of the morphological and ultrastructural characteristics of PEV and PEV-Mito.

[0042] The results are as follows Figure 1 As shown, the vesicles in the PEV-Mito group were relatively larger, while those in the PEV group were mainly smaller and lacked obvious complex internal structures. This indicates that platelets may release extracellular vesicles carrying mitochondrial-related components during activation and budding, and compared with the PEV group, the PEV-Mito group showed more obvious mitochondrial-like features in its ultrastructure.

[0043] Example 2: Detection of mitochondrial membrane potential in PEV and PEV-Mito This example aims to confirm the mitochondrial membrane potential status in PEV and PEV-Mito samples. The JC-1 probe was used to detect the mitochondrial membrane potential.

[0044] (1) Experimental methods Before the experiment, JC-1 working solution was prepared according to the kit instructions: 5 μL of JC-1 dye stock solution was added to 1 mL of JC-1 staining buffer and mixed well. PEV and PEV-Mito samples were taken separately, with a total protein content of 50 μg as the loading baseline. JC-1 working solution was added and mixed well, and the samples were incubated at 37 ℃ in the dark for 20 min. After incubation, the samples were centrifuged at 18000 × g at 4 ℃ for 60 min, the supernatant was discarded to remove unbound dye, and the samples were gently resuspended in PBS. The treated samples were then added to 96-well plates with a black transparent bottom, 100 μL per well. The green and red fluorescence intensities were detected using a multi-mode microplate reader, with the detection wavelengths set to excitation / emission wavelengths of 490 / 530 nm and 520 / 590 nm. The ratio of red fluorescence intensity to green fluorescence intensity reflected the mitochondrial membrane potential level in the samples.

[0045] (2) Experimental results like Figure 2 As shown, the JC-1 aggregate / monomer ratio in the PEV-Mito group was significantly higher than that in the PBS group and the PEV group, indicating that the mitochondria carried by PEV-Mito have intact and high membrane potential and maintain good functional activity, suggesting that they may contain mitochondrial-related components with certain functional activities.

[0046] Example 3: Spatial distribution of PEV-Mito and endogenous mitochondria in HT22 cells after PEV-Mito entry This embodiment analyzes the spatial distribution relationship between PEV-Mito and endogenous mitochondria after PEV-Mito enters HT22 cells.

[0047] (1) Experimental methods HT22 cells were labeled with a green fluorescent mitochondrial probe at 37 °C for 15 min. The green fluorescent HT22 cells were then seeded in confocal dishes, and 30 μg of red fluorescently labeled PEV-Mito was added to the cell culture system. The cells were incubated at 37 °C with 5% CO2. After incubation, the supernatant was discarded, and the cells were gently washed three times with PBS to remove any untaken free vesicles. The nuclei were then counterstained with Hoechst. After each step, the cells were washed three times with PBS. Finally, images were acquired using a HIS-SIM super-resolution microscope to obtain mitochondrial signals carried by exogenous PEV-Mito, endogenous mitochondrial signals from HT22 cells, and nuclear signals. The channels were superimposed to observe the spatial distribution of exogenous and endogenous mitochondria in the cytoplasm.

[0048] (2) Experimental results like Figure 3 As shown, after labeling endogenous mitochondria in HT22 cells and treating them with PEV-Mito, both exogenous mitochondrial-related signals from PEV-Mito and endogenous mitochondrial signals from HT22 cells could be observed simultaneously within the same cell. Both types of fluorescent signals were mainly distributed in the cytoplasm. Endogenous mitochondria mostly appeared as cord-like or short rod-like structures, while exogenous mitochondrial-related signals were mostly located around them or along similar distribution areas. Further magnification of local areas revealed that exogenous and endogenous mitochondrial signals exhibited a relatively obvious spatial proximity and local overlap in some regions. These results indicate that the mitochondrial-related components carried by PEV-Mito, after entering HT22 cells, have a certain spatial distribution association with endogenous mitochondria, suggesting that exogenous mitochondria may participate in intracellular mitochondrial network-related processes after entering the cell.

[0049] Example 4: The effect of PEV-Mito on improving mitochondrial respiratory function in neurons damaged by oxidative stress This embodiment evaluates the effect of PEV-Mito on mitochondrial oxygen consumption rate in Hemin-induced oxidative stress-damaged neurons.

[0050] (1) Experimental methods The changes in oxygen consumption rate of HT22 cells in each group were detected using a cell energy metabolism analyzer. Before the experiment, Seahorse-specific cell culture plate pretreatment, probe plate hydration, and system calibration were performed according to the instrument's requirements. HT22 cells in the logarithmic growth phase were cultured at a rate of 2 × 10⁻⁶ cells / mL. 4 Cells were seeded per well in Seahorse 96-well plates and cultured at 37 ℃ in a 5% CO2 incubator to ensure full cell adhesion. HT22 cells were then treated with 50 μM Hemin for 4 hours, followed by discarding the drug-containing culture medium. The model group was cultured in fresh complete medium for 24 hours; the Hemin+PEV group was cultured in fresh complete medium containing 30 μg / mL PEV for 24 hours; and the Hemin+PEV-Mito group was cultured in fresh complete medium containing 30 μg / mL PEV-Mito for 24 hours. Working solutions of oligomycin, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), and rotenone / Antimycin A were prepared according to the SeahorseXF Mitochondrial Stress Detection Kit instructions and added sequentially to the injection wells at final concentrations of 1.5 μM, 1.5 μM, and 0.5 μM, respectively. The OCR changes of each group of cells were recorded in real time during the detection process.

[0051] (2) Experimental results like Figure 4 As shown, Hemin treatment significantly reduced basal respiration, ATP-coupled respiration, and maximal respiratory capacity of cells; compared with the Hemin+PEV group, the OCR level of the Hemin+PEV-Mito group was significantly restored, indicating that PEV-Mito can effectively improve mitochondrial respiratory function of neurons damaged by oxidative stress, and its effect is significantly better than that of ordinary PEV.

[0052] Example 5: Inhibitory effect of PEV-Mito on ROS generation in neurons damaged by oxidative stress This embodiment verifies the regulatory effect of PEV-Mito on Hemin-induced intraneuronal ROS levels.

[0053] (1) Experimental methods Changes in mitochondrial membrane potential in HT22 cells were detected using the JC-1 fluorescent probe. Cells were divided into four groups: HT22 cells were treated with 50 μM Hemin for 4 hours, followed by discarding the drug-containing culture medium. The model group was cultured in fresh complete medium for 24 hours; the Hemin+PEV group was cultured in fresh complete medium containing 30 μg / mL PEV for 24 hours; and the Hemin+PEV-Mito group was cultured in fresh complete medium containing 30 μg / mL PEV-Mito for 24 hours. After treatment, the culture medium was discarded, and the cells were gently washed three times with PBS. Following the kit instructions, a working solution of the reactive oxygen species fluorescent probe (DCFH-DA) with a final concentration of 10 μM was prepared in serum-free medium. Cells were added to this solution and incubated at 37 ℃ and 5% CO2 for 30 min in the dark. After incubation, the cells were washed three times with PBS to remove any uninfiltrated probes, and an appropriate amount of serum-free medium was added. Subsequently, fluorescence images of each group of cells were acquired under an inverted fluorescence microscope and imaged under the same exposure parameters. ImageJ software was used to perform quantitative analysis of the fluorescence images, with the average fluorescence intensity reflecting the relative level of ROS in each group of cells.

[0054] (2) Experimental results The results are as follows Figure 5 As shown, compared to the control group, Hemin treatment significantly increased cellular ROS levels; Hemin+PEV treatment effectively reduced ROS levels; and Hemin+PEV-Mito treatment further significantly reduced ROS levels. This indicates that PEV-Mito can more effectively inhibit ROS generation in neurons damaged by oxidative stress, alleviate oxidative stress damage, and protect neuronal cells.

[0055] Example 6: The effect of PEV-Mito on the recovery of neurological function in a mouse model of cerebral hemorrhage. This embodiment evaluates the effect of PEV-Mito intervention on improving neurological deficits in mice with cerebral hemorrhage.

[0056] (1) Experimental methods The modified neurological deficit score was used to evaluate the degree of neurological function impairment in mice after cerebral hemorrhage. To analyze the repair effects of platelet vesicles from different sources on neurological function after cerebral hemorrhage, a cerebral hemorrhage model was established by injecting collagenase into the mouse brain under specific conditions using a stereotaxic device. Specifically, the mouse cerebral hemorrhage model was established using the stereotaxic injection method with type VII collagenase. The day before modeling, mice were placed in the experimental environment for acclimatization. On the day of the experiment, mice were anesthetized with isoflurane inhalation and fixed on the stereotaxic device. Hair on the top of the head was shaved, and after disinfection, the scalp was incised in the midline of the top of the skull to expose the skull and anterior fontanelle. Using the anterior fontanelle as a reference point, a hole was drilled 0.5 mm anteriorly and 2.85 mm lateral to the midline. A microsyringe was inserted vertically, slowly advancing 3.2 mm downwards from the surface of the dura mater, and held for 5 min. A solution containing 0.0375 U of type VII collagenase was slowly injected at a rate of 0.05 μL / min. After injection, the needle was left in place for 10 minutes, then slowly withdrawn, the burr hole closed, and the scalp sutured. Mice were kept at constant temperature after surgery. The groups subsequently injected with PEV or PEV-Mito (100 μL / mouse) via the tail vein served as the treatment group, while the group injected with an equal volume of PBS served as the model control group. A sham surgery group (Sham) was also included, which underwent stereotactic brain puncture without collagenase injection. To further analyze the treatment effect, the mNSS was used to assess the neurological function of mice at specific time points after model establishment and tail vein injection. The mNSS includes four aspects: motor, sensory, reflex, and balance, with a total score of 18 points. Higher scores indicate more severe neurological deficits. Assessments were performed before model establishment (day 0) and on days 1, 3, 5, 7, 14, and 30 post-surgery. All evaluations were conducted in a relatively quiet environment with stable lighting, at fixed time intervals, and the scoring process was blinded.

[0057] The motor function score ranged from 0 to 6 points, primarily based on the mouse's spontaneous activity, limb coordination, and walking stability. The sensory function score ranged from 0 to 2 points, assessed by stimulating the mouse's whiskers or forelimbs and observing its tactile and avoidance responses. The reflex function score ranged from 0 to 4 points, including auricular reflex, corneal reflex, startle reflex, and grasping reflex. The balance function score was evaluated using a balance beam test. During the test, the mouse was placed on a narrow beam approximately 1 cm wide and 50 cm long, and its walking stability and foot slippage on the beam were assessed.

[0058] (2) Experimental results like Figure 6As shown: Compared with the Sham group, the mNSS scores of mice in each ICH group were significantly increased after surgery; compared with the ICH+PBS group and the ICH+PEV group, the mNSS score of the ICH+PEV-Mito group decreased significantly over time and remained at a low level on day 30, indicating that PEV-Mito can significantly promote the recovery of neurological function after cerebral hemorrhage and is more effective than ordinary PEV.

[0059] Example 7: The effect of PEV-Mito on improving fine motor function in mice with cerebral hemorrhage This embodiment further verifies the effect of PEV-Mito on improving fine motor and sensory functions in mice with cerebral hemorrhage through forelimb placement and cornering experiments.

[0060] (1) Experimental methods a) Forelimb Placement Test: The forelimb placement test was used to evaluate changes in sensorimotor integration function in mice after cerebral hemorrhage. During the test, the mouse was supported by one hand, keeping its back straight and its hind limbs off the support surface. After the position stabilized, the right whisker area was stimulated with a cotton swab to induce a left forelimb placement response, and the number of correct left forelimb placements was recorded. Each mouse underwent 10 valid tests, and the percentage of correct left forelimb placements out of the total number of tests was used as the evaluation index. If significant struggling, positional shift, or inaccurate stimulation occurred during the test, the test was invalidated and a retest was conducted. Tests were performed before modeling (day 0) and on days 1, 3, 7, 14, and 30 post-surgery, with all tests completed at fixed time intervals. b) Angle Turning Test: The angle turning test was used to evaluate changes in sensorimotor integration function and lateral behavior in mice after cerebral hemorrhage. The experimental setup consisted of two black opaque baffles, forming an approximately 30° angle proximally and extending into a narrow channel distally. During the test, mice were placed deep within an angled area and allowed to move forward along the passage, autonomously choosing their turning direction. Each mouse completed 10 consecutive valid trials, and the number of left turns was recorded. The percentage of left turns out of the total valid trials was used as the evaluation index. If a mouse stopped, retreated, or experienced significant external interference during the test, the trial was invalidated and a retest was conducted. Tests were performed before modeling (day 0) and on days 1, 3, 5, 7, 14, and 30 post-surgery, with all tests completed at fixed time intervals.

[0061] (2) Experimental results like Figure 7 As shown: a) Forelimb placement test: The accuracy rate of left forelimb placement in the ICH+PEV-Mito group increased significantly over time, significantly better than that in the ICH+Vehicle group and the ICH+PEV group; b) Turning test: The right turning rate in the ICH+PEV-Mito group decreased significantly over time, indicating that lateral behavior was effectively corrected. These results indicate that PEV-Mito can significantly improve fine motor and sensory functions in mice with cerebral hemorrhage and promote neurological function recovery.

[0062] In summary, this invention uses platelets as raw material, and through activation induction, differential centrifugation and fractionation, membrane filtration and elution enrichment, obtains platelet-derived extracellular vesicles carrying active mitochondria, achieving efficient and stable loading of functional mitochondria (PEV Mito). The obtained PEV Mito have intact structures and uniform particle size, effectively maintaining mitochondrial membrane potential and biological activity, enabling efficient delivery and intracellular internalization to damaged neurons, and restoring mitochondrial function. The method of this invention is stable, reproducible, and suitable for large-scale preparation. The prepared PEV Mito has high delivery efficiency and excellent biocompatibility, and can be used to prepare drugs for treating cerebral hemorrhage and secondary neurological injury, showing good prospects for clinical translation.

Claims

1. A type of extracellular vesicle enriched in mitochondria, characterized in that, The vesicles are derived from extracellular vesicles formed by the in vitro activation and release of platelets, and the vesicles are enriched with mitochondria or mitochondrial-related components with complete structure and functional activity.

2. The mitochondrial-enriched extracellular vesicles according to claim 1, characterized in that, The platelets are derived from mammalian blood; the mitochondria in the vesicles have intact membrane potential, and the red / green fluorescence ratio of the mitochondrial membrane potential fluorescent probe is higher than that of extracellular vesicles from common sources, and the content of adenosine triphosphate is also higher.

3. The method for preparing mitochondrial-enriched extracellular vesicles according to claim 1 or 2, characterized in that, Includes the following steps: 1) Collect whole blood from mammals and treat it with an anticoagulant to obtain anticoagulated whole blood; 2) Centrifuge the anticoagulated whole blood from step 1) and collect the upper layer of platelet-rich plasma; 3) Add stabilizing buffer to the PRP in step 2) and mix well to inhibit non-specific platelet activation; 4) Centrifuge the PRP processed in step 3) and wash it with washing buffer to obtain purified platelet precipitate; 5) Activate the platelets from step 4) to release vesicles containing mitochondria or mitochondrial-related components; 6) The activated system from step 5) was centrifuged and membrane filtered to separate crude PEV and crude PEV-Mito, respectively. 7) The crude vesicles from step 6) were purified by ultracentrifugation to obtain the target vesicle product.

4. The method for preparing mitochondrial-enriched extracellular vesicles according to claim 3, characterized in that, In step 1), the anticoagulant is a sodium citrate solution with a mass fraction of 3%-4%, and the volume ratio of the anticoagulant to whole blood is 1:8-1:

10. After blood collection, anticoagulation is achieved by gently inverting and mixing. In step 2), the centrifugation temperature is 0-10 ℃, the centrifugation speed is 1000-2000 rpm, the centrifugation time is 10-15 min, and the upper plasma layer is collected as platelet-rich plasma.

5. The method for preparing mitochondrial-enriched extracellular vesicles according to claim 3, characterized in that, In step 3), the stabilizing buffer contains ethylenediaminetetraacetic acid and prostaglandin E1, with concentrations of 0.5-2 mM and 1-5 μM, respectively, and the volume ratio of the stabilizing buffer to PRP is 1:3–1:

10. In step 4), the washing buffer contains 0.5-2 mM EDTA and 1-5 mM benzyl sulfonyl fluoride. The centrifugation conditions are 3000-5000 rpm for 15-25 min, the washing is performed 2-4 times, and the solution is resuspended in sterile phosphate buffer.

6. The method for preparing mitochondrial-enriched extracellular vesicles according to claim 3, characterized in that, In step 5), the activation agent is thrombin, the concentration of thrombin is 0.5-2 U / mL, the activation temperature is 35-40 ℃, and the activation time is 30-90 min. In step 6), centrifugation is first performed to remove unactivated platelets and cell debris. The centrifugation conditions are 3000-5000 rpm for 15-25 min. In step 6), a sterile filter membrane with a pore size of 0.20-0.40 μm is used for separation. The filtrate is crude PEV. The portion retained by the filter membrane is recovered as PEV-Mito by backwashing with sterile PBS.

7. The method for preparing mitochondrial-enriched extracellular vesicles according to claim 3, characterized in that, In step 7), the crude PEV-Mito product is centrifuged at 10000-30000 ×g and 0-10 ℃ for 30-90 min; In step 7), after centrifugation, the supernatant is discarded and the sample is resuspended in sterile PBS. The resulting sample is stored at 0-10 °C.

8. The use of the mitochondrial-enriched extracellular vesicles according to any one of claims 1-7 in the preparation of drugs for improving mitochondrial dysfunction-related diseases.

9. The application of the mitochondrial-enriched extracellular vesicles according to claim 7 in the preparation of drugs to improve mitochondrial dysfunction-related diseases, characterized in that, The diseases mentioned include cerebral hemorrhage, brain injury, ischemia-reperfusion injury, neurodegenerative diseases, and diseases related to mitochondrial defects and deficiencies.

10. The application of the mitochondrial-enriched extracellular vesicles according to claim 7 in the preparation of drugs to improve mitochondrial dysfunction-related diseases, characterized in that, The mitochondrial-enriched extracellular vesicles can be taken up by various cell types, including neurons, immune cells, and stem cells.

Images