Mitochondrial transplantation system and its application in wound healing promotion

Abstract

The application discloses a mitochondrion transplant system, which comprises mitochondria and an apoptotic vesicle membrane, and the mitochondria are carried in the apoptotic vesicle membrane. The system composed of the mitochondria and the apoptotic vesicle membrane in the application acts on endothelial cells, improves the targeting and efficiency of mitochondrion transplantation, produces mitochondrion autophagy, reactivates the mitochondrion autophagy to remove damaged mitochondria of the endothelial cells, promotes the removal of the damaged mitochondria, restores the cell functions of the endothelial cells with damaged mitochondria, and is beneficial to promoting wound healing. The system composed of the mitochondria and the apoptotic vesicle membrane is used as an active ingredient to prepare a medicine or a medical device for promoting wound healing.

Description

Technical Field

[0001] This invention relates to an organelle delivery scheme, and more particularly to a system for performing mitochondrial transplantation, acting on endothelial cells to promote wound healing, and pharmaceuticals or medical devices made therefrom. Background Technology

[0002] Endothelial cells (ECs) play a crucial role in diabetic wound healing, with mitochondria serving as key signaling centers and metabolic regulators, directly controlling endothelial cell function. However, EC mitochondria are severely damaged under prolonged high glucose and oxidative stress, and their "mitochondrial quality control system" becomes imbalanced, manifesting as excessive division, fusion, and impaired autophagy. Damaged mitochondria cannot be cleared in time, leading to the accumulation of fragmented mitochondria, releasing more reactive oxygen species (ROS) and further damaging more mitochondria, creating a vicious cycle that ultimately impairs EC migration and angiogenesis. Therefore, targeted removal of damaged mitochondria is key to restoring EC function and promoting wound repair.

[0003] Current strategies for repairing damaged mitochondrial function in diabetic wounds include promoting mitochondrial biogenesis, alleviating oxidative stress, or supplementing with exogenous mitochondria. However, these methods primarily focus on slowing the damage process and passively repairing function, with relatively singular intervention targets. Their therapeutic efficacy is significantly limited, especially when faced with mitochondrial depletion or severe mtDNA damage.

[0004] In recent years, intercellular mitochondrial transfer has gradually attracted attention as a more proactive and broader-dimensional mechanism. This process transfers functionally intact mitochondria from healthy cells to damaged cells via tunnel nanotubes (TNTs), extracellular vesicles (EVs), or gap junctions, thereby rapidly restoring their energy metabolism capacity, regulating immune status, and reshaping their metabolic phenotype and epigenetic characteristics. This mechanism has been proven to play a crucial role in the repair process of various diseases. However, in the environment of tissue damage, especially diabetic wounds, factors such as calcium overload, reactive oxygen species, and surges in pro-inflammatory and inflammatory factors can significantly inhibit mitochondrial activity and interfere with its transcellular transfer process. These damaging factors not only weaken the activity of donor cell mitochondria but also affect the formation of transfer structures such as tunnel nanotubes (TNTs), limiting the effective transport of mitochondria. Recent research in Nature found that after exogenous mitochondria enter recipient cells, they do not directly integrate into their mitochondrial network but tend to co-localize with autophagosomes, activating the PINK1–Parkin-dependent mitophagy pathway. This process is considered an important mechanism for cells to screen and evaluate the function of heterologous mitochondria. If this pathway is blocked, the therapeutic effect of mitochondrial transfer is hindered by the persistent presence of mitochondrial dysfunction, which not only weakens mitochondrial stability but also affects its sustained support for the metabolic function of recipient cells. Notably, high glucose and metabolic toxin stimulation in the microenvironment of diabetic wounds have been shown to severely impair mitophagy function, leading to rapid intracellular degradation of exogenous mitochondria and difficulty in maintaining their function. Therefore, while promoting mitochondrial transfer, how to rebuild the homeostasis of the autophagy system and ensure the metabolic integration and functional performance of transplanted mitochondria has become a key breakthrough in overcoming the bottleneck in the treatment of diabetic wounds.

[0005] Mitophagy-mediated dysfunction or the elimination of excess mitochondria plays a crucial role in numerous processes, including inflammation, metabolic shifts, and cellular reprogramming. Therefore, cells have evolved multiple pathways to ensure timely, rapid, and precise activation of mitophagy in response to various stimuli. However, impaired mitophagy leads to mitochondrial dysfunction and decreased cell viability, necessitating exogenous interventions to restore tissue regeneration. Currently, common strategies for restoring autophagy include mitochondrial uncoupling agents, mitochondrial membrane stabilizers, and gene editing methods. However, mitochondrial uncoupling agents and mitochondrial membrane stabilizers often suffer from high toxicity and difficulty in dosage control; gene therapy, on the other hand, has limitations such as short mRNA expression duration, poor stability, susceptibility to degradation by in vitro and in vivo nucleases, and the potential for unmodified mRNA to trigger innate immune responses, requiring repeated administration to maintain efficacy. Therefore, there is a need to explore safer, more direct, and more durable alternatives for stimulating autophagy. Summary of the Invention

[0006] One object of the present invention is to provide a mitochondrial transplantation system that improves the targeting and efficiency of mitochondrial transplantation.

[0007] Another objective of this invention is to provide a mitochondrial transplantation system that facilitates the clinical application of mitochondrial transplantation.

[0008] Another objective of this invention is to provide a mitochondrial transplantation system that facilitates the action of mitochondria on endothelial cells, thereby promoting wound healing.

[0009] Another object of the present invention is to provide a mitochondrial transplantation system for use in the preparation of pharmaceuticals or medical devices for treating diabetic wounds.

[0010] Unlike highly metabolic cells such as cardiomyocytes and alveolar epithelial cells, endothelial cells (ECs) often integrate transplanted mitochondria into their own mitochondrial networks to support oxidative phosphorylation (OXPHOS). Endothelial cells have relatively low mitochondrial abundance and primarily function as signal regulators rather than energy metabolism centers.

[0011] Verification showed that mitochondrial transplantation did not increase the overall mitochondrial content, but rather activated PINK1-Parkin-mediated mitophagy. Under chronic oxidative damage conditions (with the continuous accumulation of dysfunctional mitochondria), mitochondrial transplantation preferentially triggered a quality control response, promoting the clearance of damaged mitochondria. This clearance process subsequently promoted cellular function recovery through downstream protective pathways (rather than enhanced energy metabolism). In addition to restoring mitophagy, endothelial cells treated with mitochondrial transplantation showed enhanced glycolysis.

[0012] A mitochondrial transplantation system includes mitochondria and an apoptotic vesicle membrane (AVM), with the mitochondria carried within the apoptotic vesicle membrane.

[0013] Another mitochondrial transplantation system includes mitochondria and apoptotic vesicle membranes, measured by protein concentration, with apoptotic vesicle membranes and free mitochondria combined in a 2:1 ratio.

[0014] Another mitochondrial transplantation system consists of mitochondria and apoptotic vesicle membranes. The mitochondria are carried within the apoptotic vesicle membranes, and the apoptotic vesicle membranes and free mitochondria are combined in a 2:1 ratio based on protein concentration.

[0015] The mitochondrial transplantation system of the present invention has a particle size of less than 1 micrometer, for example, 800nm±100nm, and a zeta potential of -11mV±1mV.

[0016] AVMs possess biologically known "eat-me" signaling molecules, enabling endothelial cells to specifically recognize and actively take them up.

[0017] AVMs are prepared by endothelial cell apoptosis, for example, by treating the cells with 5 μM astrone for 12 hours.

[0018] The system of this invention, composed of mitochondria and apoptotic vesicle membranes, acts on endothelial cells, improving the targeting and efficiency of mitochondrial transplantation. This induces mitophagy, reactivating it to clear damaged mitochondria from endothelial cells, promoting the clearance of damaged mitochondria, restoring cellular function of mitochondria-damaged endothelial cells, and facilitating wound healing. Using this system of mitochondria and apoptotic vesicle membranes as the active ingredient, drugs or medical devices that promote wound healing can be formulated.

[0019] These pharmaceutical excipients can be those commonly used in various formulations, such as, but not limited to, isotonic agents, buffers, excipients, fillers, binders, and lubricants; or they can be selected for compatibility with the substances in the formulation, such as emulsifiers (e.g., Tweeen-80, Pluronic, and Poloxamer), solubilizers, antibacterial agents, pH adjusters, analgesics, and antioxidants. These excipients can effectively improve the stability and solubility of the compounds contained in the composition or change the release and absorption rates of the compounds, thereby improving the metabolism of various compounds in the body and enhancing the drug delivery effect of the composition.

[0020] In aqueous injections, excipients generally include isotonic agents and buffer solutions, as well as necessary emulsifiers (such as Tweeen-80, Pluronic, and Poloxamer), solubilizers, and antibacterial agents. In addition, they may include other pharmaceutically acceptable excipients, such as antioxidants, pH adjusters, and analgesics.

[0021] Excipients used in the preparation of solid dosage forms generally include fillers (such as starch, powdered sugar, dextrin, lactose, compressible starch, microcrystalline cellulose, calcium sulfate, dicalcium phosphate, and mannitol), binders (such as ethanol, starch paste, sodium carboxymethyl cellulose, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, hydroxypropyl methyl cellulose, gelatin solution, sucrose solution, and aqueous or alcoholic solutions of polyvinylpyrrolidone), disintegrants (such as dry starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, croscarmellose, and croscarmellose sodium), and lubricants (such as magnesium stearate, micronized silica gel, talc, hydrogenated vegetable oil, polyethylene glycol 4,000, polyethylene glycol 6,000, and magnesium lauryl sulfate).

[0022] The excipients used in the preparation of emulsions are generally water, oil (such as fatty acids), emulsifiers, and necessary preservatives and flavoring agents.

[0023] Various excipients and compounds are used to formulate dosage forms that facilitate drug delivery, such as, but not limited to, aqueous injections, powder injections, pills, powders, tablets, patches, suppositories, emulsions, creams, gels, granules, capsules, aerosols, sprays, powder inhalers, sustained-release formulations, and controlled-release formulations. Furthermore, excipients may be used to achieve specific drug delivery purposes or methods, such as sustained-release, controlled-release, and pulsatile administration, including, but not limited to, gelatin, albumin, chitosan, polyethers, and polyester polymers, such as, but not limited to, polyethylene glycol, polyurethane, polycarbonate, and their copolymers. The main manifestations of "facilitating drug delivery" include, but are not limited to, improved therapeutic efficacy, increased bioavailability, reduced toxicity and side effects, and improved patient compliance.

[0024] Drug-containing medical devices combining drugs and medical devices are already quite common, such as wound dressings containing a system of mitochondria and apoptotic vesicle membranes as active ingredients. The system of mitochondria and apoptotic vesicle membranes in this invention is also loaded or coated onto scaffold materials as an active ingredient for the manufacture of medical devices for diabetic wound repair. Common scaffold materials include collagen, hyaluronic acid, chitosan, PVA, PLA, PLGA, PGA, and metals. These materials can also be mixed with biocompatible biodegradable materials to form microneedles and microneedle arrays, or loaded into metal microneedles to create microneedle chips. When the microneedles pierce the skin, they release the system of mitochondria and apoptotic vesicle membranes, accelerating the healing of (diabetic) wounds.

[0025] A method for preparing the mitochondrial transplantation system of the present invention includes:

[0026] Mix apoptotic vesicle membranes with free mitochondria (e.g., at a protein concentration ratio of 2:1), and treat the mixture with gentle intermittent sonication (e.g., maintaining 4°C, sonicating for 20 seconds each time, stopping for 10 seconds, and repeating this cycle) for 5 minutes; centrifuge at 3500±100×g (e.g., 4°C, 15 minutes), remove the supernatant, and wash to obtain the final product.

[0027] A gel system for mitochondrial transplantation includes hyaluronic acid, polyvinyl alcohol, and a mitochondrial transplantation system, wherein the hyaluronic acid and polyvinyl alcohol are cross-linked to form a matrix encapsulating the mitochondrial transplantation system, and the mitochondrial transplantation system is released in a sustained manner.

[0028] In the gel system of this invention, the ratio of hyaluronic acid to polyvinyl alcohol is 1:3 by weight. Hyaluronic acid-phenylboronic acid derivatives are preferred.

[0029] The gel system of the present invention contains a mitochondrial transplantation system with a protein concentration of 5 µg / mL.

[0030] The method for preparing the gel system of the present invention includes:

[0031] Hyaluronic acid-phenylboronic acid (HA-PBA, 20 mg / mL) and polyvinyl alcohol (PVA, 60 mg / mL) were dissolved separately in a system containing a mitochondrial-apoptotic vesicle membrane complex (Mito-AVM). The two solutions were vortexed in equal volumes for 20 seconds to initiate a cross-linking reaction, resulting in a mitochondrial-apoptotic vesicle membrane-hydroxyphenylboronic acid-polyvinyl alcohol hydrogel (denoted as: Mito-AVM@HPP).

[0032] The technical solutions provided by this invention optimize mitochondrial transplantation protocols at the cellular, tissue, and spatiotemporal levels, facilitating the clinical application of mitochondrial transplantation. At the cellular level, this invention bioengineers mitochondria. Previous mitochondrial delivery strategies have largely employed cationic liposomes or cell membrane encapsulation techniques to prevent extracellular degradation of mitochondria. However, these systems have significant functional limitations: cationic liposomes bind to mitochondria via electrostatic interactions and achieve cell delivery through membrane fusion, but they lack inherent targeting specificity, have poor delivery efficiency, and exhibit potential biotoxicity; cell membrane-based encapsulation techniques, while possessing natural homing capabilities, are too large relative to mitochondria, physically affecting delivery efficiency. In contrast, apoptotic vesicle membranes (AVMs) are an optimized carrier that integrates the advantages of the aforementioned two systems while overcoming their respective shortcomings: AVMs have excellent size compatibility with mitochondria, enabling efficient encapsulation. Furthermore, AVMs carry natural "eat-me" signals, promoting specific recognition and active uptake by endothelial cells through a mechanism similar to burial. AVMs not only possess mitochondrial protective capabilities, but also exhibit approximately 150% higher targeting and transplantation efficiency in vivo compared to free mitochondria, making them a highly promising mitochondrial transplantation therapy vector.

[0033] Regarding optimization at the tissue and spatiotemporal levels, this invention proposes a hydrogel (e.g., HPP hydrogel) as a solution for the unique microenvironment of diabetic wounds. Hydrogels are ideal materials for wound healing, as they can both close the wound and absorb secreted tissue fluid, thereby promoting healing. This HPP hydrogel possesses excellent physical properties and can be easily injected into the wound using a syringe. Furthermore, the hydrogel can responsively degrade and release the mitochondrial-apoptotic vesicle membrane complex (Mito-AVM), exerting a therapeutic effect in the high-glucose, high-ROS environment of diabetic wounds. These properties give this hydrogel significant advantages and are beneficial for the clinical application of MT (tumor thrombosis).

[0034] The technical solution of this invention combines organelle-level bioengineering with the application of biomaterials, which is beneficial for clinical application. It not only provides a new solution for the treatment of chronic wounds in diabetic patients, but also provides a universal solution for mitochondrial transplantation therapy for diseases characterized by mitochondrial dysfunction. Attached Figure Description

[0035] Figure 1 The images show the validation results of mitochondrial transplantation (MT) enhancing EC function. A represents the tube formation experiment, B represents the migration experiment, C represents the number of connection points in each experimental group (angiogenesis), D represents the total segment length in each experimental group, E represents the total pore area in each experimental group, F represents the relative area of ​​migrated cells in each experimental group, G represents the volcano plot of differentially expressed metabolites, H represents the bubble chart of KEGG pathway differential abundance (DA) scores, I represents the KEGG classification diagram, J represents the relative levels of glucuronic acid in each experimental group, K represents the relative levels of pyruvate in each experimental group, L represents the relative levels of adrenaline in each experimental group, M represents the relative levels of coenzyme Q2 in each experimental group, N represents the relative levels of genipin in each experimental group, and O represents the relative levels of S-adenosylmethionine in each experimental group.

[0036] Figure 2This figure shows the validation results of MT's ability to enhance angiogenesis in endothelial cells (ECs) by clearing damaged mitochondria; where A represents the Western blot results of autophagy markers (LC3-II / I, p62) and mitophagy markers (PINK1, Parkin, TOM20) in endothelial cells from the control group and the mitochondrial transplantation group (MT-ECs). The results of the blotting (β-actin as an internal reference protein) are shown in Figure B. B shows transmission electron microscopy (TEM) images of each experimental group. In the control group, swollen mitochondria (marked with blue circles) are present in the endothelial cells, and mitochondrial cristae show breakage and fragmentation (marked with blue arrows). In the mitochondrial transplantation group, autophagolysosomes (marked with red arrows) and mitochondrial autophagosomes (marked with red circles, representing the double-membrane structure surrounding mitochondria) are visible in the endothelial cells. C shows confocal microscopy images of LC3 (green) and TOM20 (red) in each experimental group. D shows confocal microscopy images of lysosomes (green) and mitochondria (red) in each experimental group. E shows the detection results of MitoSO Red fluorescence (representing mitochondrial reactive oxygen species mtROS) in live endothelial cells of each experimental group. F shows the quantitative analysis of cellular reactive oxygen species (DCFH-DA labeling) and mitochondrial membrane potential (JC-1 Red / The representative pseudo-color scatter plot of green fluorescence ratio, G is the result of lumen formation experiment of each experimental group, H is the statistical plot of mitochondrial reactive oxygen species level of each experimental group, I is the statistical plot of cellular reactive oxygen species (ROS) level of each experimental group, J is the statistical plot of mitochondrial membrane potential (expressed as JC-1 fluorescence ratio) of each experimental group, and K is the statistical plot of angiogenesis capacity (expressed as the number of connection points) of each experimental group.

[0037] Figure 3Figure 1 shows the preparation and characterization results of the mitochondrial-apoptotic vesicle membrane complex (Mito-AVM). A is a schematic diagram of the Mito-AVM preparation process; B is a Western blot diagram of endothelial cells (ECs), apoptotic vesicles (ApoVs), and apoptotic vesicle membranes (AVMs) for detecting endothelial cell markers (CD31), apoptosis indicators (caspase-3 and cleaved caspase-3), and content markers (histone H3) (β-actin was used as an internal reference protein); C is a quantitative diagram of histone H3 in apoptotic vesicles (ApoVs) and apoptotic vesicle membranes (AVMs); D is a diagram of DiO2-labeled apoptotic vesicle membranes (AVMs, green) and DsRed. The diagram shows the fluorescence co-localization field of view of the labeled mitochondria (red). E is the transmission electron microscope (TEM) field of view of apoptotic vesicles (ApoVs), apoptotic vesicle membranes (AVMs), mitochondria, and the mitochondrial-apoptotic vesicle membrane complex (Mito-AVM). F is the statistical diagram of the hydrodynamic diameter distribution measured by dynamic light scattering (DLS) for each experimental group. G is the zeta potential measurement result for each experimental group. H is the confocal microscope field of view of each experimental group after co-incubation for 4 hours. I is the flow cytometry quantitative analysis result of mitochondrial uptake for each experimental group. J is the in vivo tracking immunofluorescence field of view of tissue sections 24 hours after subcutaneous injection into diabetic wounds for each experimental group. K is the quantitative statistical diagram of mitochondrial uptake for each experimental group. L is the statistical diagram of in vivo experiment (fluorescence intensity per unit of CD31 positive area) for each experimental group.

[0038] Figure 4The following graphs show the results of Mito-AVM's promotion of damaged mitochondrial clearance and restoration of cell function. Specifically, A shows the live / dead staining results of endothelial cells (ECs) after 24 hours of treatment with Mito-AVM at a ratio of 100:1; B shows the quantitative analysis results of live / dead cells; C shows the relative ATP levels of each experimental group; D shows the cell proliferation kinetics detected by CCK-8 assay within 72 hours when the Mito-AVM to endothelial cell ratio is 100:1; E shows the scratch healing experiment results for each experimental group; F shows the lumen formation experiment results for each experimental group; G shows the wound closure rate (%gap closure) statistics for each experimental group; H shows the number of vascular connection points for each experimental group; I shows the total segment length statistics for each experimental group; J shows the mesh area statistics for each experimental group; K shows the mesh number statistics for each experimental group; and L shows the results of DCFH-DA assay after 48 hours of treatment for each experimental group. The results of the detection of cellular reactive oxygen species (ROS) levels are shown in the figure. M is the statistical graph of ROS fluorescence intensity in each experimental group, N is the statistical graph of JC-1 aggregate / monomer ratio in each experimental group, and O is the fluorescence image of JC-1 in each experimental group (red: aggregate; green: monomer).

[0039] Figure 5Figure 1 shows the preparation, characterization, and functional verification results of mitochondrial-apoptotic vesicle membrane-hydroxyphenylboronic acid-polyvinyl alcohol hydrogel (Mito-AVM@HPP). Figure 2 shows the process flow diagram for preparing Mito-AVM@HPP hydrogel via boric acid-diol crosslinking; Figure 3 shows a bright-field photograph of the gelation process of HPP hydrogel and Mito-AVM@HPP hydrogel; Figure 4 shows a scanning electron microscope (SEM) field of view of HPP hydrogel; Figure 5 shows a SEM field of view of Mito-AVM@HPP hydrogel; Figure 6 shows the experimental curves of storage modulus (G′) and loss modulus (G″) of HPP hydrogel; and Figure 7 shows the results of Mito-AVM@HPP hydrogel preparation, characterization, and functional verification. Experimental curves of hydrogel storage modulus (G′) and loss modulus (G″), G is a bright-field photograph of the macroscopic adhesion of the hydrogel on human skin (standing posture), H is the viscosity test curve of each group of hydrogels, I is a photograph of the self-healing ability of segmented hydrogels, J is the result of step strain test of Mito-AVM@HPP hydrogel at 37℃ (repeated application of 100% strain for 60 seconds and 1000% strain for 60 seconds), K is a photograph of hydrogel extruded through a 21G needle, L is a photograph of hydrogel precisely forming the "SJTU" pattern, M is a photograph of hydrogel completely degraded after 7 days in an environment containing 100 μM hydrogen peroxide (H2O2) and 50 mM glucose, N is the modulus test curve of hydrogel after 72 hours of exposure to 100 μM H2O2, and O is the modulus test curve of hydrogel after 72 hours of exposure to 50 mM glucose. The modulus test curve after hours, P is the statistical graph of the modulus test of hydrogel, Q is the live / dead staining result of endothelial cells after 24 hours of contact with hydrogel, R is the bioluminescence tracking result of retention in each experimental group, and S is the curve of change of fluorescence intensity of wound in each experimental group.

[0040] Figure 6Figure 1 shows the effects of various treatments on the healing of diabetic wounds. A represents wound photographs of the control group (Control), apoptotic vesicle membrane group (AVM), mitochondrial transplantation group (MT), mitochondrial-apoptotic vesicle membrane complex group (Mito-AVM), HPP hydrogel group (HPP), and mitochondrial-apoptotic vesicle membrane-hydroxyphenylboronic acid-polyvinyl alcohol hydrogel group (Mito-AVM@HPP) at 0, 3, 7, 14, and 21 days after treatment. B represents the quantitative statistical graph of wound closure rate in each experimental group at 3 days after treatment. C represents the quantitative statistical graph of wound closure rate in each experimental group at 7 days after treatment. D represents the quantitative statistical graph of wound closure rate in each experimental group at 14 days after treatment. E represents the quantitative statistical graph of wound closure rate in each experimental group at 21 days after treatment. F represents the H&E staining of wounds in each experimental group. G represents the statistical graph of neovascularization in each experimental group. H represents the Masson staining of wounds in each experimental group. Trichrome staining diagram: I is a statistical diagram of the relative collagen deposition of animals in each experimental group 7 days after treatment, and J is a statistical diagram of the relative collagen deposition of animals in each experimental group 21 days after treatment.

[0041] Figure 7 The images show the results of different treatments on angiogenesis, vascular maturation, and collagen deposition 7 days after treatment. A is an immunofluorescence image of CD31, B is a statistical graph of the relative expression level of CD31, C is an immunofluorescence image of α-smooth muscle actin, D is a statistical graph of the relative expression level of α-SMA, E is an immunofluorescence image of type I collagen (COL-I), F is a statistical graph of the relative expression level of type I collagen, G is an immunofluorescence image of type III collagen (COL-III), and H is a statistical graph of the relative expression level of type III collagen. Detailed Implementation

[0042] The technical solution of the present invention will be described in detail below with reference to the accompanying drawings. The embodiments of the present invention are only used to illustrate the technical solution of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solution of the invention without departing from the spirit and scope of the technical solution of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention.

[0043] The specific experimental methods used in the following embodiments of the present invention are described below:

[0044] 1) Cell Culture

[0045] Human umbilical vein endothelial cells (HUVECs; purchased from Shanghai Anwei Biotechnology Co., Ltd., China), hereinafter referred to as endothelial cells (ECs), were cultured at 37°C using endothelial cell medium (ScienCell, catalog number 1001). All experiments used endothelial cells from passages 6 to 12. To simulate mitochondrial damage, endothelial cells were treated with 100 μM hydrogen peroxide (H2O2) for 24 hours before the start of the experiments.

[0046] 2) Mitochondrial mitoDsRed labeling

[0047] The pLV-CMV-MCS-mitoDsRed-Puro plasmid (Chinese Miaoling plasmid) was transfected into endothelial cells (ECs) to prepare DsRed-labeled mitochondria. After transfection, the mitochondria could be clearly observed under a fluorescence microscope.

[0048] 3) Mitochondrial isolation and mitochondrial transplantation

[0049] Mitochondria were isolated from donor cells using a cell mitochondrial isolation kit (Beyotime Biotechnology, China, catalog number C3601). The specific steps are as follows:

[0050] 1. Digest the cells in the culture dish with trypsin, wash twice with pre-cooled PBS, and then gently resuspend them in mitochondrial separation reagent supplemented with benzyl sulfonyl fluoride (PMSF);

[0051] 2. After incubating at 4°C for 20 minutes, homogenize the cell suspension 40 times;

[0052] 3. Centrifuge the homogenate at 4℃ and 1000×g for 10 minutes, and collect the supernatant;

[0053] 4. The supernatant was centrifuged at 4℃ and 3500×g for 15 minutes to obtain the isolated mitochondria.

[0054] The morphology of free mitochondria can be clearly observed by staining the isolated mitochondrial precipitate with mitochondrial tracer (MitoTracker); at the same time, the functional activity of mitochondria can be verified by JC-1 staining.

[0055] Mitochondrial transplantation (MT) was performed as follows: An appropriate amount of isolated mitochondria (or mitochondrial-apoptotic vesicle membrane complex Mito-AVM / mitochondrial-apoptotic vesicle membrane-hydroxyphenylboronic acid-polyvinyl alcohol hydrogel Mito-AVM@HPP) were added to the culture medium and co-incubated with endothelial cells under standard culture conditions. Twelve hours after transplantation, the endothelial cells were washed three times with PBS, and the culture medium (ECM) was replaced with fresh medium for continued culture.

[0056] 4) Live / dead staining method for detecting cell viability

[0057] Cell viability was assessed 24 hours after mitochondrial transplantation using a live / dead staining kit (Sigma-Aldrich, catalog number 04511). Cells were incubated with diluted dye at 37°C for 30 minutes, followed by observation and photography using a fluorescence microscope. The fluorescence intensity and positive staining area of ​​each experimental group were quantitatively analyzed using ImageJ software (National Institutes of Health, NIH).

[0058] 5) CCK-8 assay

[0059] Add CCK-8 reagent (Beyotime Biotechnology, catalog number C0038) to a 96-well plate containing cells, incubate for 30-45 minutes, and then measure the absorbance at 450 nm using a microplate reader (Thermo Fisher Scientific, model Varioskan Flash 3001).

[0060] 6) Transfer experiment

[0061] The migration ability of endothelial cells was assessed using a standard scratch healing assay, and the specific steps are as follows:

[0062] Endothelial cells were cultured in 6-well plates. Once the cell confluence reached 80%-90%, a straight line was drawn along the center of the well plate using a 1000 μL pipette tip. Mitochondrial transplantation and other treatments were then performed immediately.

[0063] Take photos at 0 hours, 24 hours, and 48 hours after treatment to observe the wound healing process;

[0064] Using ImageJ software (National Institutes of Health, NIH), the migration capacity of endothelial cells at each time point was quantitatively analyzed by comparing the "remaining wound area / initial wound area".

[0065] 7) Angiogenesis experiment

[0066] The following experimental protocol was used to assess the angiogenesis capacity of endothelial cells, and the specific steps are as follows:

[0067] Thaw the matrix gel (Corning, catalog number 356234) overnight at 4°C to prepare pre-cooled 24-well culture plates;

[0068] Add 50 μL of matrix gel to each well and incubate at 37°C for 60 minutes in a cell culture incubator to allow it to solidify;

[0069] Cells treated with different enzymes were digested with trypsin, resuspended in complete culture medium, and the cell concentration was adjusted to 5 × 10⁶ cells / mL. 5 cells / mL;

[0070] Carefully add 200 μL of cell suspension to each well coated with matrix gel, incubate for 6 hours, remove the culture plate, and observe the formation of tubular structures using a phase contrast microscope;

[0071] The angiogenesis analysis plugin (Angiogenesis Analyzer) of ImageJ software (National Institutes of Health, USA) was used to quantitatively analyze the network structure by measuring the number of connection points, total segment length, total network area, and number of network pores.

[0072] 8) Standardized detection of cellular ATP levels

[0073] Cellular ATP levels were detected using an ATP assay kit (Beyotime Biotechnology, catalog number S0026). The simplified steps are as follows: cells were treated with ATP lysis buffer for 30 minutes, followed by the addition of assay solution, and the luminescence intensity of each well was measured using a microplate reader. Protein concentration was determined using the Bradford method, and the ATP levels were standardized accordingly.

[0074] 9) Analysis of the effects of mitochondrial transplantation on the cellular metabolome

[0075] To assess the effect of mitochondrial transplantation on mitochondrial-damaged endothelial cells, cells were first treated with 100 μM hydrogen peroxide (H2O2) for 24 hours to induce oxidative damage, followed by mitochondrial transplantation at a ratio of 100:1. After 72 hours, Shanghai Lumin Biotechnology Co., Ltd. of China was commissioned to conduct comprehensive metabolomics analysis on the two groups of cells using liquid chromatography-mass spectrometry (LC-MS / MS) and gas chromatography-mass spectrometry (GC-MS / MS) platforms. The specific analysis procedures and data statistics methods were consistent with our previous studies

[33] .

[0076] 10) Western blot analysis

[0077] Endothelial cells in a 10cm culture dish were washed three times with PBS, digested with trypsin, collected by centrifugation, and washed three more times with PBS.

[0078] Lyse cells with RIPA lysis buffer (Thermo Fisher Scientific) containing phosphatase inhibitors and protease inhibitors (Roche Applied Sciences), and incubate the lysis buffer on ice for 20-30 minutes;

[0079] Centrifuge at 4℃ and 14000×g for 15 minutes and collect the supernatant (protein sample).

[0080] Dilute the protein sample with 4×Laemmli buffer (Bio-Rad, catalog number 1610747) containing 10% β-mercaptoethanol and denature at 95°C for 5 minutes.

[0081] 25 μg of protein was loaded onto each lane, and the protein was separated by SDS-PAGE gel electrophoresis. The protein was then transferred to a polyvinylidene fluoride (PVDF) membrane (Sigma-Aldrich).

[0082] The PVDF membrane was blocked at room temperature for 10 minutes using a rapid blocking solution (Beyotime Biotechnology, catalog number P0240), followed by co-incubation with the primary antibody.

[0083] Wash the PVDF membrane three times with TBST buffer for 10 minutes each time, then incubate it with the corresponding secondary antibody at room temperature for 1 hour, and then wash it three times with TBST buffer.

[0084] The protein bands were observed and photographed using ECL chemiluminescent solution (Proteintech).

[0085] The primary antibodies used included: anti-LC3 antibody (Proteintech, catalog number 14600-1-AP, dilution 1:1000), anti-P62 / SQSTM1 antibody (Proteintech, catalog number 18420-1-AP, dilution 1:1000), anti-PINK1 antibody (Proteintech, catalog number 23274-1-AP, dilution 1:1000), anti-Parkin antibody (Proteintech, catalog number 14060-1-Ap, dilution 1:1000), anti-TOM20 antibody (Proteintech, catalog number 11802-1-AP, dilution 1:1000), anti-β-actin antibody (Proteintech, catalog number 20536-1-AP, dilution 1:2000), and anti-CD31 antibody. Antibody (Proteintech, USA, catalog number 66065-2-Ig, dilution ratio 1:5000), anti-Caspase3 antibody (Affinity, catalog number AF6311, dilution ratio 1:1000), cleaved-Caspase3 antibody (CST, catalog number 9664T, dilution ratio 1:1000), anti-histone H3 antibody (Abcam, catalog number Ab201456, dilution ratio 1:2000).

[0086] 11) Mitochondrial and lysosomal live-cell imaging

[0087] Following mitochondrial transplantation, endothelial cells in glass-bottomed culture dishes (NiceBio, catalog number 801002) were incubated for 48 hours, followed by live cell staining.

[0088] Stain with 200 nM mitochondrial tracer Red CMXRos (Beyotime Biotechnology, catalog number C1035) at 37°C for 20 minutes, then wash with PBS;

[0089] Stain with 500 nM lysosomal tracer Green DND-26 (Thermo Fisher Scientific, catalog number L7526) at 37°C for 30 minutes, and finally wash with PBS;

[0090] Imaging was performed in complete culture medium using a Zeiss LSM 800 confocal microscope (40x objective, sequential scanning mode, 488nm / 587nm laser), with minimal light exposure during the process.

[0091] 12) Detection of mitochondrial reactive oxygen species (mtROS)

[0092] The mitochondrial superoxide anion level was detected using the mitochondrial superoxide anion fluorescent probe MitoSO™ Red (Beyotime Biotechnology, catalog number S0061S). Cells in 6-well plates were treated under experimental conditions for 48 hours, washed twice with PBS, and then incubated with 2.5 μM MitoSO™ Red working solution at 37°C for 30 minutes. After removing the staining solution and washing twice more with PBS, the generation of mitochondrial superoxide anions was immediately observed and quantified using a fluorescence microscope.

[0093] 13) Detection of reactive oxygen species (ROS) in cells

[0094] Endothelial cells were directly stained using a 10 μM DCFH-DA probe (Reactive Oxygen Spectrometry Kit, Beyotime Biotechnology, catalog number S0033S): After incubation at 37°C for 15 minutes and washing three times with PBS, the reactive oxygen species levels in the cells were immediately analyzed using a fluorescence microscope and flow cytometry (FITC channel).

[0095] 14) JC-1 staining detection

[0096] Mitochondrial membrane potential was assessed using JC-1 staining (Beyotime Biotechnology, catalog number C2006). Cells were incubated with JC-1 staining solution at 37°C for 20 minutes, washed twice with JC-1 dilution buffer, and the red / green fluorescence ratio was measured by microscopy and flow cytometry (FITC / PE channel was used after trypsin digestion of cells) to quantitatively analyze mitochondrial membrane potential depolarization.

[0097] 15) TMRE staining detection

[0098] Follow the instructions in the kit (TMRE kit, Beyotime Biotechnology, catalog number C2001S): stain cells with TMRE in complete culture medium (incubate at 37°C in the dark for 30 minutes), wash three times with pre-warmed PBS, and observe immediately under a fluorescence microscope.

[0099] 16) Immunofluorescence staining

[0100] Cells were fixed with 4% paraformaldehyde (PFA) for 15 minutes, permeabilized with 0.1% Triton X-100 / PBS for 10 minutes, and blocked with 5% normal serum for 1 hour.

[0101] The primary antibody was co-incubated with glass-cultured cells at 4°C overnight;

[0102] Remove the primary antibody, wash three times with PBS, and then incubate the sample with the corresponding secondary antibody at room temperature for 1 hour;

[0103] Wash three times with PBS, mount with anti-fluorescence quenching mounting medium, and store at 4°C in the dark.

[0104] 17) Isolation and characterization of the mitochondrial-apoptotic vesicle membrane complex (Mito-AVM)

[0105] Preparation and isolation of apoptotic vesicles (ApoVs): Endothelial cells cultured under standard conditions (37℃, 5% CO2) were treated with 5 μM astrosporin (STS; Sigma-Aldrich) for 12 hours to generate apoptotic vesicles. The generation of apoptotic vesicles was confirmed by observing characteristic morphological changes (nuclear membrane rupture, membrane bubbling) under an optical microscope. After digesting the cells with trypsin, the cells were centrifuged at 300×g for 10 minutes. The supernatant was then centrifuged again at 3000×g for 10 minutes to isolate the apoptotic vesicles. The apoptotic vesicles were dissolved in PBS and stored at -80℃.

[0106] Preparation of apoptotic vesicle membranes (AVMs): Apoptotic vesicles were resuspended in hypotonic lysis buffer (10 mM Tris, pH 7.4; 10 mM MgCl2; 1 mM PMSF) and incubated at 4 °C for 1 hour. The membranes were then sonicated for 10 minutes using an ultrasonic cleaner (KQ2200E), centrifuged at 10000×g for 10 minutes, and washed three times with PBS to obtain purified apoptotic vesicle membranes.

[0107] Characterization of apoptotic vesicle membranes: Protein concentration was determined using the BCA protein quantification kit (Absin, catalog number abs9232); particle size and zeta potential were detected using a dynamic light scattering instrument (Malvin, Zetasizer Nano ZSE); and the expression of CD31, Caspase3, cleaved Caspase3, and histone H3 was detected by Western blotting to verify the identity and purity of the apoptotic vesicle membranes.

[0108] Preparation and observation of mitochondrial-apoptotic vesicle membrane complex (Mito-AVM): Apoptotic vesicle membranes and free mitochondria were mixed at a protein concentration ratio of 2:1, and the mixture was subjected to mild intermittent sonication for 5 minutes. After centrifugation at 3500×g for 15 minutes at 4℃, the supernatant was removed, and the mixture was washed three times with pre-cooled PBS. The particle size and zeta potential were measured by dynamic light scattering. Before fusion, the apoptotic vesicle membranes were labeled with DiO2 dye, and the mitochondria were labeled with DsRed. The specific structure of the mitochondrial-apoptotic vesicle membrane complex was observed by confocal microscopy.

[0109] 18) Preparation of mitochondrial-apoptotic vesicle membrane-hydroxyphenylboronic acid-polyvinyl alcohol hydrogel (Mito-AVM@HPP)

[0110] Hyaluronic acid-phenylboronic acid (HA-PBA) conjugates were prepared (Advanced materials (Deerfield Beach, Fla. 2024;36(19):e2311964), and the successful conjugation was verified by ¹H nuclear magnetic resonance (¹H NMR) in deuterated water (D2O).

[0111] Hyaluronic acid-phenylboronic acid (HA-PBA, 20 mg / mL) and polyvinyl alcohol (PVA; Aladdin, CAS No.: 9002-89-5, 60 mg / mL) were dissolved separately in deionized water containing mitochondrial-apoptotic vesicle membrane complex (Mito-AVM) at a volume ratio of 1:1. The two solutions were vortexed in equal volumes for 20 seconds to initiate a cross-linking reaction, resulting in mitochondrial-apoptotic vesicle membrane-hydroxyphenylboronic acid-polyvinyl alcohol hydrogel (Mito-AVM@HPP).

[0112] 19) Characterization of mitochondrial-apoptotic vesicle membrane-hydroxyphenylboronic acid-polyvinyl alcohol hydrogel (Mito-AVM@HPP)

[0113] Microstructure observation: After freeze-drying the hydrogel, its cross-section was photographed using a scanning electron microscope (Hitachi, SU3500) to observe the microstructure;

[0114] Rheological testing: Rheological tests were performed using a rotational rheometer equipped with a 20mm parallel plate. For each test, 400μL of HPP hydrogel or Mito-AVM@HPP hydrogel was taken and placed on a 37℃ temperature-controlled platform. The tests included:

[0115] Oscillatory strain scan: frequency 10 rad / s, strain sequence is 100% strain for 60 seconds, 1000% strain for 60 seconds, repeated 3 times;

[0116] Viscosity curves: The viscosity was monitored by gradually increasing the shear rate from 0 to 1000 s⁻¹ to evaluate the shear thinning behavior and structural recovery properties of the hydrogel.

[0117] 20) Diabetic rat wound healing model

[0118] Female Sprague Dawley rats (weighing 180-220g) were intraperitoneally injected with streptozotocin (STZ, 50mg / kg) to induce diabetes. One week later, the successful establishment of the diabetes model was confirmed by a tail vein blood glucose level ≥16.7mmol / L.

[0119] Diabetic rats were randomly divided into 6 groups (n=4 per group): control group, apoptotic vesicle membrane group (AVM), mitochondrial group (Mito), mitochondrial-apoptotic vesicle membrane complex group (Mito-AVM), HPP hydrogel group (HPP), and mitochondrial-apoptotic vesicle membrane-hydroxyphenylboronic acid-polyvinyl alcohol hydrogel group (Mito-AVM@HPP).

[0120] Under sodium pentobarbital anesthesia, the hair on the back of the rat was removed, and four full-thickness circular wounds (10 mm in diameter) were made using a sterile biopsy punch.

[0121] Wound healing was tracked using standardized digital photography at 0, 3, 7, 14, and 21 days after wound formation, and the wound closure rate (percentage reduction in area relative to day 0) was calculated using ImageJ software.

[0122] 21) In vivo optical imaging

[0123] After successfully establishing a diabetic wound model, DiD-labeled mitochondrial-apoptotic vesicle membrane complex (Mito-AVM) was injected subcutaneously around the wound, and DiD-labeled mitochondrial-apoptotic vesicle membrane-hydroxyphenylboronic acid-polyvinyl alcohol hydrogel (Mito-AVM@HPP) was applied topically to the wound surface. At 1, 2, and 3 days post-administration, fluorescence intensity was longitudinally monitored using an IVISSpectrum imaging system (Xenogen, USA) with standardized acquisition parameters (excitation / emission wavelength: 640nm / 670nm). The signal intensity (photons / sec / cm² / sr) of preset regions of interest (ROIs) was quantitatively analyzed using Living Image software to compare the tissue retention kinetics of the two administration methods.

[0124] 22) Histological and immunofluorescence analysis of wound tissue

[0125] Wound tissue was collected at 7 and 21 days after wound formation, fixed with 4% paraformaldehyde for 48 hours, and then embedded in paraffin and sectioned (7 μm thick).

[0126] Histological assessment: Hematoxylin and eosin (H&E) staining was used to observe vascular structures (identified by lumen filled with erythrocytes), and Masson trichrome staining was used to observe collagen deposition;

[0127] Immunofluorescence analysis: After dewaxing, paraffin sections were antigen-retrieved using Tris-EDTA buffer or citrate buffer, followed by blocking with 5%-10% serum. The sections were then co-incubated overnight at 4°C with anti-CD31 antibody (Abcam, catalog number ab182981, dilution 1:400), anti-α-smooth muscle actin (α-SMA) antibody (HUABIO, catalog number ET1607-53, dilution 1:1000), anti-type I collagen (COL-I) antibody (HUABIO, catalog number HA722517, dilution 1:200), or anti-type III collagen (COL-III) antibody (HUABIO, catalog number HA720050, dilution 1:200). After incubation with fluorophore-conjugated secondary antibody (dilution 1:200), the nuclei were stained with DAPI. Finally, the fluorescence signal was quantitatively analyzed using ImageJ software.

[0128] 23) Statistical analysis

[0129] No data exclusion criteria were set during the analysis, and quantitative data were expressed as mean ± standard deviation (mean ± SD). Unpaired two-tailed t-tests (pairwise comparisons) or one-way ANOVA were used for comparisons between groups, and Tukey's test (using GraphPad Prism v10.3.1 software) was used for comparisons among multiple groups. Statistical significance thresholds were set as follows: *p < 0.05, **p < 0.01, ***p < 0.001.

[0130] Example 1: Verification of MT-enhanced EC activity and function

[0131] Mitochondrial damage was induced in endothelial cells (ECs) by treating them with 100 μM H₂O₂ for 24 h to verify whether MT could salvage the function of ECs with mitochondrial damage. This pretreatment placed endothelial cells in an oxidative environment that mimicked the extracellular hydrogen peroxide (H₂O₂) levels measured in chronic diabetic wounds. This condition effectively induced mitochondrial damage and cell dysfunction while avoiding excessive apoptosis (Antioxid Redox Signal. 2006; 8 (3-4):243-70).

[0132] Mitochondria derived from healthy endothelial cells were added to damaged endothelial cells at different mitochondrial-to-cell ratios (control group, 10:1, 30:1, 100:1, 250:1, 500:1) to verify the repair potential of mitochondrial transplantation (MT) on mitochondrial-damaged endothelial cells. After 24 hours, compared with the control group, the 100:1 mitochondrial transplantation ratio significantly improved endothelial cell viability, increased normalized ATP levels, improved angiogenesis (Figure 1A, CE), and enhanced migration ability (Figure 1B, F).

[0133] Notably, although most studies on mitochondrial transplantation suggest that the cytoprotective effect stems from enhanced oxidative phosphorylation (OXPHOS), our metabolomics analysis of endothelial cells from the mitochondrial transplantation group (Mito group) and the untreated control group showed no increase in oxidative phosphorylation activity—consistent with the fact that endothelial cells primarily rely on glycolysis for energy. Instead, we identified 92 differentially expressed metabolites (Figure 1G, Figures S1C-D), and pathway analysis revealed significant upregulation of pentose / glucuronide tautomerism (represented by glucuronide), glycolysis (represented by pyruvate), and the adrenergic signaling pathway (represented by adrenaline) (Figure 1H, JL).

[0134] Furthermore, mitochondrial transplantation significantly increased the levels of autophagy-related metabolites (Figure 1I), specifically by decreasing levels of coenzyme Q2 (which promotes electron transport chain function) and genipin (which inhibits uncoupling protein 2 (UCP2)) (Figure 1M-N). Previous studies have shown that these compounds can promote mitophagy activation. Simultaneously, the decrease in S-adenosylmethionine (SAM) levels was associated with a significant enhancement of autophagic flux (Figure 1O).

[0135] In summary, these findings suggest that mitochondrial transplantation (MT) does not repair endothelial cell (EC) function by enhancing oxidative phosphorylation (OXPHOS), but rather by inducing a metabolic shift towards glycolysis dominance and activating mitophagy—these potential mechanisms collectively enhance the vitality and function of ROS-damaged endothelial cells.

[0136] Example 2: MT enhances EC angiogenesis by clearing damaged mitochondria through mitophagy.

[0137] Western blot analysis showed that, compared with the control group, the LC3-II / I ratio of endothelial cells in the mitochondrial transplantation group (Mito group) was increased and the p62 level was decreased, indicating enhanced autophagic flux (Figure 2A). Furthermore, the significant upregulation of PINK1 and Parkin, and the decreased expression of TOM20, provided direct evidence for the activation of mitophagy. Figure 2 A).

[0138] Transmission electron microscopy (TEM) revealed significant mitochondrial swelling and cristae breakage in the control group endothelial cells, typical characteristics of ROS-induced damage. Conversely, the mitochondrial transplantation group endothelial cells exhibited abundant autolysosomes and intact double-membrane autophagosomes encapsulating mitochondrial remnants (Figure 2B). Immunofluorescence staining results were consistent with these findings: LC3 colocalization with mitochondria was increased in the mitochondrial transplantation group endothelial cells (Figure 2C), with a Pearson correlation coefficient (R value) of 0.35, significantly higher than the 0.17 in the control group endothelial cells (Figure S1E). Furthermore, in the fluorescence co-staining experiment using mitochondrial tracer and lysosomal tracer, the lysosomal fluorescence intensity was higher in the mitochondrial transplantation group endothelial cells compared to the control group, and the correlation between lysosomes and mitochondria was stronger. Figure 2 D). The above results indicate that mitochondrial transplantation can simultaneously enhance endothelial cell autophagic flux and mitophagy activation.

[0139] Using pharmacological methods, autophagy was inhibited with 5 mM 3-methyladenine (3-MA) ​​to clarify the role of enhanced mitophagy after mitochondrial transplantation in "clearing damaged mitochondria and improving endothelial cell function." CCK-8 assays showed that autophagy inhibition led to decreased endothelial cell viability and significantly weakened the therapeutic effect of mitochondrial transplantation, indicating that autophagy plays a crucial role in mediating the efficacy of mitochondrial transplantation. Similarly, autophagy inhibition also significantly reduced ATP levels in endothelial cells, suggesting that autophagy itself is an important energy source for these cells. Furthermore, although mitochondrial transplantation treatment significantly increased pyruvate kinase levels (confirming enhanced glycolysis), autophagy inhibition did not result in a statistically significant decrease in its activity, suggesting that glycolytic flux may be regulated through complex signaling pathways beyond autophagy.

[0140] TMRE staining results showed that mitochondrial transplantation increased mitochondrial membrane potential (ΔΨm) by 32%, but this increase almost completely disappeared after the addition of 3-MA. Similarly, the mitochondrial transplantation-mediated decrease in mitochondrial reactive oxygen species (mtROS) and cellular reactive oxygen species (ROS) levels, as well as the improvement in the JC-1 aggregate / monomer ratio, were all significantly weakened after autophagy inhibition (Fig. 2E, F, HJ). These results indicate that mitochondrial transplantation-activated mitophagy can selectively clear damaged mitochondria, thereby improving overall mitochondrial membrane potential and cellular mitochondrial homeostasis. Subsequently, we obtained consistent results through lumen formation experiments. Figure 2 G, K).

[0141] Mechanistically, mitochondrial transplantation clears dysfunctional mitochondria through mitophagy—these mitochondria are the main source of excessive reactive oxygen species (ROS) production under oxidative stress. This process can terminate the release of pathological ROS, restore mitochondrial homeostasis, thereby enhancing the angiogenesis capacity of endothelial cells and ultimately forming a self-reinforcing cycle of vascular repair.

[0142] Example 3: Preparation and Characterization of Mito-AVM

[0143] Apoptotic vesicle membranes (AVMs) derived from HUVECs were selected for mitochondrial membrane fusion, enhancing their ability to target ECs and be phagocytosed by ECs. Figure 3 A). First, apoptosis was induced by STS, and ApoVs were extracted by differential centrifugation. Their contents were further removed using an engineered process of hypotonic-ultrasonic-centrifugation to obtain apoptotic vesicle membranes (AVMs). Western blotting validated the characterization of ApoVs and AVMs, demonstrating that engineered AVMs (using Histone H3 as a representative) had their contents removed. Figure 3 B, C). Next, by sonicating and co-incubating DiO-stained AVMs with mitochondria extracted from healthy HUVECs transfected with Mito-DsRed at a ratio of 2:1, followed by centrifugation, Mito-AVMs fused with mitochondria were obtained. Fluorescence co-localization demonstrated that the fusion efficiency was close to 95%, and the structure of the AVM encapsulating the mitochondria could be clearly observed. Figure 3 D). TEM revealed the distinctive mitochondrial crest in the cross-section of the Mito-AVM, along with significant thickening of part of its outer membrane accompanied by membrane fusion. Figure 3 E).

[0144] The physical properties of Mito-AVM were detected by DLS and zeta potential, and the particle size was found to be approximately 800 nm. Figure 3 F), the zeta potential is approximately -11mV ( Figure 3 G). Meanwhile, when exposed to oxidative stress with 100 µmol / L H₂O₂, Mito-AVM exhibited stronger stability than Mito, indicating a protective effect of AVM against Mito. Then, by co-culturing free Mito and Mito-AVM with ECs for 4 h, it was found that ECs had a significantly higher uptake efficiency of Mito-AVM. Flow cytometry analysis of the difference revealed that the mitochondrial transplantation efficiency of Mito-AVM was nearly twice that of free Mito. Figure 3 H, I, K). In a diabetic rat wound model, equal amounts of free Mito and Mito-AVM were subcutaneously injected. After 24 hours, it was found that the mitochondrial transplantation efficiency of endothelial cells in the Mito-AVM group was significantly higher. Figure 3 J, L). Finally, we subcutaneously injected AVM, Mito, and Mito-AVM into the wounds of diabetic rats, respectively. After 48 hours, we collected samples for Western blotting experiments and found that the Mito-AVM group expressed the strongest mitophagy, confirming that Mito-AVM had the highest mitochondrial transplantation efficiency.

[0145] Example 4: Validation of Mito-AVM's ability to promote the clearance of damaged mitochondria and restore cellular function in damaged ECs.

[0146] Co-culturing AVMs with ECs at different ratios and verifying their activity changes using the CCK-8 assay revealed that the optimal concentration of AVMs was 1 µg / ml. Based on this, co-culturing with different Mito-AVM donor / recipient ECs ratios and live / dead assays showed that, while maintaining the optimal AVM concentration, cell viability was significantly enhanced when the Mito-AVM to recipient ECs ratio ranged from 10:1 to 250:1. Figure 4 A and Figure 4 B), and the number of dead cells increases when the ratio reaches 500:1, which is related to apoptosis caused by excessive autophagy.

[0147] ATP levels in ECs treated with different concentrations of Mito-AVM were measured, and it was found that after reaching a certain amount of transplanted Mito, the ATP levels in ECs remained relatively stable. Figure 4 C). The CCK-8 experiment also demonstrated that ECs treated with Mito-AVM at a concentration of 100:1 exhibited excellent proliferative capacity within 72 hours. Figure 4 D). Therefore, subsequent experiments were conducted at a ratio of 100:1.

[0148] Cell function and intracellular mitochondrial status were verified by dividing the cell into control group, AVM, Mito, and Mito-AVM groups. Scratch and tube formation experiments were also conducted. Figure 4 In E and F groups, the Mito-AVM group showed the best cell migration ability. Figure 4 G) and pipe forming capacity ( Figure 4 HK). Detection of ROS levels and mitochondrial membrane potential levels within ECs showed that, after 48 h of treatment, the ROS level in the Mito-AVM group was significantly lower than that in other groups ( Figure 4 L, Figure 4 Similarly, its mitochondrial membrane potential was significantly better than other groups (M). Figure 4 N、 Figure 4Finally, we subcutaneously injected AVM, Mito, and Mito-AVM into the wounds of diabetic rats, and collected samples for Western blot analysis after 48 hours. The results showed that the Mito-AVM group exhibited the strongest mitophagy.

[0149] These findings collectively demonstrate that Mito-AVMs, compared to free mitochondria, have a stronger ability to promote the clearance of damaged mitochondria from ROS-damaged ECs and enhance cellular function.

[0150] Example 5: Mito-AVM@HPP Hydrogel Controlled Release System

[0151] A hydrogel adapted for diabetic wounds to improve the situation of excessively rapid local drug clearance. First, PBA molecules were grafted onto the HA backbone via amide bonds. Successful synthesis of HA-PBA was confirmed by hydrogen nuclear magnetic resonance. 20 mg / ml of HA-PBA and 60 mg / ml of PVA were mixed homogeneously in a 1:1 volume ratio, and the mixture rapidly cross-linked and gelled through the formation of borate ester bonds. Figure 5 B). Scanning electron microscopy (SEM) observation of the cross-section of the lyophilized hydrogel showed that both HPP and Mito-AVM@HPP hydrogels formed porous network structures, and the distribution of Mito-AVM in Mito-AVM@HPP hydrogels (red) was also observed. Figure 5 C, D).

[0152] The mechanical properties of HPP and Mito-AVM@HPP hydrogels were characterized using rheological measurements. As shown by the strain sweep test, both HPP and Mito-AVM@HPP hydrogels maintained their gel-like viscoelastic state within a stress range of 1–100%. However, as the strain increased to 1000%, G′ decreased and became smaller than G′′, indicating the shear thinning properties of the hydrogels. Figure 5 E, F). Viscosity tests and skin adhesion experiments demonstrated that the hydrogel has good adhesive properties, making it very suitable for application in skin environments. Figure 5 G, H).

[0153] By cutting the hydrogel into two pieces to evaluate its self-healing properties and injectability, it was found that the boundaries between the two small pieces of hydrogel became blurred and they merged together to form a single, monolithic hydrogel. Figure 5 I). Simultaneously, a step-strain test (strain = 100–1000%) was used for three cycles. When the strain increased to 1000%, the gel network rapidly ruptured, and G′ and G'' decreased rapidly. However, after the strain decreased, G′ and G'' quickly recovered, indicating that the hydrogel has good self-healing ability. Figure 5 J). Then we filled a 1ml syringe with the green-dyed Mito-AVM@HPP hydrogel and injected it into water using a 21G syringe needle. Figure 5 K and Figure 5 L), and the letters "SJTU" (representing "Shanghai Jiao Tong University") were injected into the surface of the petri dish. Figure 5 K) indicates its good injectability.

[0154] Because borate ester bonds are highly sensitive to the presence of ROS and glucose, the borate ester hydrogel network may be disrupted in applications to diabetic wounds. The ROS / glucose dual-responsive degradation of the hydrogel was confirmed using 100 µM H₂O₂ and 50 µM glucose solutions, respectively. After 7 days of treatment, almost all Mito-AVM@HPP hydrogels were observed to completely self-degrade into liquid. Figure 5 M). Meanwhile, a strain sweep test showed that Mito-AVM@HPP hydrogels exposed to 100 µM H2O2 and 50 µM glucose solution for 3 days exhibited significantly reduced G' and G'' compared to the control. Figure 5 (NP). This indicates that the Mito-AVM@HPP hydrogel has good ROS and sugar-responsive degradation capabilities.

[0155] ECs were co-cultured with HPP and Mito-AVM@HPP hydrogels for 24 h. Live / dead staining showed that not only did the number of dead cells in the two hydrogel treatment groups not increase, but the number and activity of ECs significantly increased after treatment with Mito-AVM@HPP hydrogel, confirming its good biocompatibility. Figure 5 Q). We then evaluated the sustained release of DiD-labeled Mito-AVM by in vivo imaging. In a diabetic mouse dorsal wound model, approximately 60% of the Mito-AVM injected intradermally around the wound was cleared at 24 h, and almost completely cleared at 72 h. However, after topical application of Mito-AVM@HPP hydrogel to the wound surface, the Mito-AVM clearance rate remained relatively stable, and it exhibited a significantly longer retention time at the wound site. This demonstrates that Mito-AVM@HPP hydrogel can achieve sustained release of Mito-AVM. Figure 5 R, S).

[0156] Example 6: Histochemical Validation of Mito-AVM@HPP Hydrogel in Promoting Healing of Diabetic Wounds

[0157] Animals with a diabetic wound model were selected and observed and photographed on days 0, 3, 7, 14, and 21 after drug administration. Furthermore, rats were sacrificed at the early stage of wound healing (day 7) and the late stage of healing (day 21), and histological staining was performed, including HE staining and Masson's trichrome staining. Figure 6 As shown in Figure A, compared with the control group, the other five experimental groups all showed varying degrees of stronger wound healing, with the Mito-AVM@HPP group showing the best wound healing effect at days 7, 14, and 21.

[0158] ImageJ software quantitative analysis of the relative wound area percentage in each group showed that, compared with the control, the residual wound area in the AVM, Mito, Mito-AVM, and Mito-AVM@HPP groups was significantly smaller on days 7 and 14. Specifically, the wound area in the Mito-AVM group was significantly smaller than that in the AVM and Mito groups, and the wound area in the Mito-AVM@HPP group was significantly smaller than that in the Mito-AVM group, achieving the best treatment effect. Figure 6 (BE) This indicates that Mito-AVM@HPP has the strongest ability to accelerate the healing of diabetic wounds.

[0159] HE staining results of diabetic wounds as follows Figure 6 As shown in F. On day 7, the wound length in the control group was the longest compared to the other 5 groups, while the wound length in the Mito-AVM@HPP group was the shortest. The number of new blood vessels in each group showed that the number of new blood vessels in the AVM, Mito, Mito-AVM, and Mito-AVM@HPP groups was significantly higher than that in the control group, while the number in the Mito-AVM@HPP group was significantly higher than that in the AVM, Mito, and Mito-AVM groups. Figure 6 (G), which shows a prognosis favorable for diabetic wound healing. On day 21, since the Mito, Mito-AVM, and Mito-AVM@HPP groups all tended to complete healing, the differences were not significant, but the Mito-AVM@HPP group also showed the best tissue maturity.

[0160] Masson tricolor dyeing as Figure 6 H showed that, compared with the control group, the AVM, Mito, Mito-AVM, and Mito-AVM@HPP groups all showed significantly more collagen (blue) deposition (H). Figure 6 I). On day 21, collagen deposition in the Mito-AVM@HPP group was significantly higher than in all other groups ( Figure 6 J).

[0161] In summary, mitochondrial transplantation endows chronic wounds with the ability to vascularize and deposit collagen. More importantly, with the modification of AVM and the integration of HPP, Mito-AVM@HPP achieves EC-targeted and highly efficient sustainable mitochondrial transplantation, thus demonstrating optimal ability to promote chronic wound healing.

[0162] Example 7: Validation of biomarkers for promoting wound healing in diabetic patients using Mito-AVM@HPP hydrogel

[0163] Immunofluorescence staining was performed on EC marker CD31, smooth muscle cell markers α-SMA, COL1, and COL3 to verify angiogenesis and collagen deposition in diabetic wounds. Figure 7 As shown.

[0164] On day 7, the control group had fewer cells and almost no angiogenesis, while the Mito, Mito-AVM, and Mito-AVM@HPP groups had more angiogenesis. Figure 7 A), and CD31 expression was significantly increased in both the Mito-AVM and Mito-AVM@HPP groups, with the Mito-AVM@HPP group showing the highest CD31 expression ( Figure 7 B). α-SMA immunofluorescence staining also showed similar results ( Figure 7 (C, D). This indicates that Mito-AVM@HPP can promote endothelial cell proliferation and angiogenesis to accelerate the healing of diabetic wounds. In immunofluorescence staining of COL-I and COL-III, compared to the control group, the expression of COL-I in the AVM, Mito-AVM, and Mito-AVM@HPP groups was significantly increased, and the expression of COL-III in the Mito, Mito-AVM, and Mito-AVM@HPP groups was significantly increased. Therefore, this suggests that Mito-AVM@HPP can significantly increase the synthesis of COL-I and COL-III to enhance collagen deposition capacity.

[0165] The immunofluorescence staining results on day 21 showed a similar trend to those on day 7. In the early stages of wound healing, COL-III played a dominant role in tissue repair, while in the later stages, COL-I dominated, supporting tissue strength. On day 21, the COL-I / COL-III ratio was the highest among the six groups in the Mito-AVM@HPP group. (The relative ratio of COL-I to COL-III is more important in assessing collagen deposition capacity.)

[0166] Therefore, it can be concluded that Mito-AVM@HPP can significantly promote collagen deposition.

Claims

1. A mitochondrial transplantation system for promoting wound healing, characterized in that... It includes mitochondria and apoptotic vesicle membranes. Mitochondria are contained within apoptotic vesicle membranes, which are produced by the apoptosis of endothelial cells.

2. The mitochondrial transplantation system according to claim 1, characterized in that, Based on protein concentration, apoptotic vesicle membranes and free mitochondria were combined in a 2:1 ratio.

3. The mitochondrial transplantation system according to claim 1, characterized in that... The particle size is less than 1 micrometer.

4. The mitochondrial transplantation system according to claim 1, characterized in that... Prepare it as follows: The apoptotic vesicle membrane was mixed with free mitochondria, and the mixture was subjected to gentle intermittent sonication for 5 minutes. After centrifugation at 3500±100×g, the supernatant was removed, and the mixture was washed to obtain the final product.

5. The use of the mitochondrial transplantation system according to claim 1 in the manufacture of pharmaceuticals or medical devices that promote wound healing.

6. The application according to claim 5, characterized in that... The mitochondrial transplantation system described herein acts on endothelial cells, reactivating mitophagy to clear damaged mitochondria from endothelial cells.

7. The application according to claim 5, characterized in that... The mitochondrial transplantation system is encapsulated within a hydrogel.

8. The application according to claim 7, characterized in that... The hydrogel comprises hyaluronic acid and polyvinyl alcohol.

9. The application according to claim 7, characterized in that... The hydrogel comprises hyaluronic acid-phenylboronic acid, which is crosslinked with polyvinyl alcohol to form a gel.

10. The application according to claim 7, characterized in that... The hydrogel contains a mitochondrial transplantation system with a protein concentration of 5 µg / mL.

Images