Delivery method of mesenchymal stem cell mitochondria based on outer vesicle wrapping

Abstract

The invention provides a method for delivering mesenchymal stem cell mitochondria based on outer vesicle wrapping, and belongs to the field of stem cell delivery. The delivery system is composed of hybrid vesicles fixed with curcumin-sulfydryl derivatives and a mitochondrial preservation solution wrapped by the hybrid vesicles. The hybrid vesicle is composed of a mesenchymal stem cell membrane and a functionalized liposome; the dosage of the functionalized liposome is greater than or equal to that of the mesenchymal stem cell membrane. According to the mitochondrial delivery system provided by the invention, immune escape is realized through the mesenchymal stem cell membrane, and the in-vivo circulation time is prolonged; precise mitochondrial targeting is achieved through TPP modification. According to the invention, the curcumin-sulfydryl derivative is covalently anchored to the vesicle membrane, so that a dynamic anti-oxidation barrier is formed, whole-course protection is provided for mitochondria, membrane fusion is remarkably promoted, and the delivery efficiency is improved. Concentration-dependent toxicity and functional interference possibly caused by direct dissolution of curcumin in a preserving fluid are avoided, and synergism of protection and delivery is realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of stem cell delivery, in particular to a delivery method of mesenchymal stem cell mitochondria based on exovesicle packaging. BACKGROUND

[0002] Mitochondria, as a multifunctional organelle in cells, has long been known as the "power station" of cells, mainly responsible for energy production, apoptosis and other key life processes. In recent years, with the in-depth study of mitochondrial function, more and more evidence shows that extracellular mitochondria can be endocytosed by cells, enter the interior of cells in an intact form and exert biological functions. This discovery has laid the foundation for mitochondrial therapy, a new type of biological therapy using active mitochondria as a therapeutic means. Mitochondrial-related diseases cover a wide range of human health problems, including neurodegenerative diseases, mental illnesses, tumors, cardiovascular diseases and diabetes, etc. Although these diseases manifest in different tissues and exhibit different symptoms, mitochondrial dysfunction is a common feature, mainly manifested as insufficient energy production, increased reactive oxygen species and abnormal apoptosis signals due to impaired oxidative phosphorylation.

[0003] Traditional treatment strategies for mitochondrial-related diseases mainly focus on small molecule drugs, vitamin and coenzyme supplementation, etc., aiming to improve mitochondrial function or alleviate oxidative stress damage. However, in the development process of most diseases, the structure and function of mitochondria often appear irreversible damage or irreparable damage, so the effect of drugs targeting the repair of cell's own mitochondrial function is limited. In this context, the direct introduction of healthy and functionally complete mitochondria into cells is considered as an effective method to fundamentally treat mitochondrial diseases. Studies have shown that the behavior of exogenous mitochondria after entering the recipient cells exhibits environmental response characteristics: when mitochondria enter cells in a physiological environment, they can improve cell energy supply and promote cell survival; however, when mitochondria enter hypoxic and acidic tumor tissues, they will produce a large number of oxygen free radicals and induce cell death. This unique pharmacological property makes mitochondrial therapy not only useful for restoring damaged tissue function, but also capable of selectively eliminating tumor cells.

[0004] Although mitochondrial therapy shows broad application prospects, in actual application, the preservation, transportation and in vivo delivery of active mitochondria face multiple technical challenges. Mitochondria are organelles that are extremely sensitive to the microenvironment, and their functional stability is significantly affected by oxygen content, pH, temperature and other factors. Mitochondria that have been separated from the original cell environment are extremely susceptible to loss of activity under in vitro conditions, which poses a serious challenge to the storage and use of mitochondrial drugs. Studies have shown that the stability of mitochondrial membrane potential, oxidative phosphorylation function and structural integrity are very difficult to maintain in vitro, requiring highly specialized preservation media and condition control.

[0005] In terms of delivery, mitochondria, as micro-scale organelles, face many physiological and biological barriers in their targeted transport in vivo. Directly injected naked mitochondria are easily cleared in the circulatory system and difficult to accumulate effectively in target tissues. At the same time, the complex physiological environment in vivo can cause mitochondria to degrade or lose function before reaching the target cells. Even if mitochondria can reach the vicinity of target cells, the process of entering cells is also full of efficiency problems. Studies have shown that the main mechanism of mitochondria entering cells may be the actin-dependent endocytosis pathway, but the efficiency of this process is limited by many factors and varies significantly between different cell types.

[0006] Mitochondrial delivery strategies based on extracellular vesicles are one of the promising solutions that have emerged in recent years. Extracellular vesicles are nanoscale vesicles with membrane structure secreted by cells naturally, and play an important role in intercellular communication. Studies have shown that a class of large-sized extracellular vesicles, microvesicles, can transport intact mitochondria to adjacent cells, helping to restore the function of mitochondria in recipient cells. At the same time, small extracellular vesicles have also been shown to be able to transport mitochondrial components such as proteins or DNA for extracellular vesicle-based disease treatment.

[0007] Using vesicles to deliver mitochondria has multiple significant advantages. First, vesicles, as natural nanocarrier systems, have good biocompatibility and low immunogenicity in vivo, and can effectively protect their contents from degradation. Encapsulating mitochondria inside vesicles can create a microenvironment similar to the original cell, which is conducive to the maintenance of mitochondrial function. Studies have shown that through a precise controlled preparation process, up to 71.7% of vesicles can contain active mitochondria without nuclear material, ensuring the safety and effectiveness of treatment.

[0008] Second, the vesicle structure itself has excellent cell affinity, which can promote efficient endocytosis of the contents by target cells. Microvesicles loaded with mitochondria can be efficiently endocytosed by mesenchymal stem cells and deliver active mitochondria to mesenchymal stem cells, improving the mitochondrial network structure and function of mesenchymal stem cells. This high efficiency of delivery has been verified in various disease models, especially in the treatment of diabetes combined with myocardial infarction, where vesicle-delivered mitochondria can comprehensively improve the mitochondrial function of mesenchymal stem cells, thereby effectively regulating the phenotype of mesenchymal stem cells.

[0009] However, the technology of vesicle-delivered mitochondria is still in its development stage and faces several important limitations. The standardization of vesicle production is the first challenge. Currently, there is a lack of more standardized and unified methods for the extraction and identification of different types of extracellular vesicles, resulting in significant differences between batches and making it difficult to meet the consistency and repeatability requirements for clinical applications. At the same time, the cargo loading efficiency of vesicles still needs to be improved, especially for large-sized organelles such as mitochondria, and current techniques are still difficult to achieve efficient and stable encapsulation.

[0010] Another key issue is the limited targeting ability of natural vesicles. Although engineering can improve the targeting of vesicles, the introduction of exogenous components can increase the complexity and unpredictability of the system. In in vivo applications, the uncertain distribution and rapid clearance of vesicles also hinder their drug delivery efficiency, which needs to be further optimized to improve the accumulation in target tissues.

[0011] Live mitochondria have solid scientific basis and broad application prospects for the treatment of mitochondrial-related diseases, but they still face many challenges in the actual clinical application. The vesicle delivery system provides a powerful tool to solve these challenges, but the technology itself still needs to make further breakthroughs in standardization, efficiency and precision, in order to realize its clinical translation potential. SUMMARY

[0012] The first object of the present application is to provide a mitochondrial delivery system consisting of hybrid vesicles with curcumin-thiol derivatives fixed thereon and a mitochondrial preservation solution wrapped by the hybrid vesicles; The hybrid vesicles consist of mesenchymal stem cell membranes and functional liposomes; and the amount of functional liposomes is greater than or equal to that of mesenchymal stem cell membranes; The functional liposomes at least include the following components: DOPC, ≥ 50 mol% Cholesterol, ≥ 20 mol% DSPE-PEG2000, ≥ 2 mol% DSPE-PEG2000-TPP, ≥ 0.2 mol% DSPE-PEG2000-Mal, ≥ 0.5 mol%.

[0013] Preferably, the average diameter of the hybrid vesicles before wrapping mitochondria is ≥ 200 nm.

[0014] Preferably, the preparation method of the curcumin-thiol derivative is to activate the phenolic hydroxyl group of curcumin, then to undergo amidation reaction with the thiol compound of amino group to generate curcumin-thiol derivative crude product; and then to purify to obtain curcumin-thiol derivative.

[0015] The preparation method of the curcumin-thiol derivative includes the following steps: S1 curcumin activation: dissolve curcumin in anhydrous dimethylformamide, cool to ≤ 5℃ in a water bath; add N,N'-dicyclohexyl carbodiimide and 4-dimethylaminopyridine in sequence, stir the reaction to generate curcumin activation intermediate; S2 amidation reaction: the preparation method of the curcumin-thiol derivative is to activate the phenolic hydroxyl group on the curcumin, then to carry out amidation reaction with 2-mercaptoethylamine free amine to generate curcumin-thiol derivative crude product; then purification is carried out to obtain curcumin-thiol derivative. The amine is dissolved in anhydrous dimethylformamide, and is added dropwise into the reaction system of S1 to obtain curcumin-thiol derivative crude product solution; S3 post-treatment and purification: pour the curcumin-thiol derivative crude product solution into ice ether, filter to collect the filtrate; remove the solution by rotary evaporation, dissolve the residue in ethyl acetate, then wash with citric acid solution, sodium bicarbonate solution and sodium chloride solution in turn; dry the organic phase, filter and rotary evaporate to obtain the crude product; After column chromatography purification of the crude product, curcumin-thiol derivative is obtained.

[0016] Preferably, the mesenchymal stem cell membrane of the hybrid vesicle is obtained by lysing, homogenizing, differential centrifugation purification and washing of the cultured mesenchymal stem cells; The functionalized liposome of the hybrid vesicle is obtained by mixing the components, rotary evaporation into a film, hydration, nano-membrane extrusion and purification; The hybrid vesicle is obtained by mixing the mesenchymal stem cell membrane and the functionalized liposome in a buffer, hybridizing by microfluidic technology, and then gradient centrifuging and resuspending.

[0017] Preferably, the curcumin-thiol derivative is covalently bonded to the hybrid vesicle through a thiol-maleimide bond; it comprises the following steps: S1 pretreatment: according to the required target surface density of maleimide, calculate the feeding amount of each component; weigh the required hybrid vesicle and curcumin-thiol derivative solution; S2 click chemistry reaction: dilute the hybrid vesicle with buffer to the target particle concentration, add the corresponding amount of curcumin-thiol derivative solution, and carry out click chemistry reaction in the dark; S2 click chemistry reaction: dilute the hybrid vesicle with buffer to the target particle concentration, add the corresponding amount of curcumin-thiol derivative solution, and carry out click chemistry reaction in the dark under nitrogen protection; S3 purification and post-treatment: after the reaction is completed, use a size exclusion chromatography column for purification to remove unreacted curcumin derivative and solvent, and collect the component containing the hybrid vesicle with curcumin-thiol derivative fixed; In the step S1 pretreatment: The surface density σ = ([Mal]total x N a ) / (N ves x πd 2 ); wherein [Mal]total is the total concentration of maleimide in the hybrid vesicle suspension; N a is the Avogadro constant; N ves is the vesicle particle concentration; π is the constant of the circle; d is the average diameter of the hybrid vesicle; The surface density σ is 5-15 / μm 2 .

[0018] Preferably, the hybrid vesicle with the immobilized curcumin-mercapto derivative is encapsulated in a mitochondrial preservation solution by microfluidic encapsulation. The pH of the mitochondrial preservation solution is 7.4, and at least contains the following components: sucrose, 250mM; mannitol, 50mM; HEPES, 10mM; KH2PO4, 5mM; MgCl2, 5mM; EDTA, 0.5mM; succinate, 5mM; pyruvate, 2mM; malate, 1mM; BSA, 0.1% (W / v); protease inhibitors, 1×; The 1× protease inhibitor refers to the corresponding 1-fold effective working concentration.

[0019] Preferably, the mitochondria are infiltrated in the mitochondrial preservation solution, and the mitochondria are derived from mesenchymal stem cells.

[0020] The second object of the present application is to provide a mitochondrial preparation, wherein the mitochondria are encapsulated by the aforementioned mitochondrial delivery system.

[0021] The third object of the present application is to provide the use of the aforementioned mitochondrial preparation in the preparation of a drug or health product related to mitochondrial dysfunction.

[0022] The mitochondrial delivery system prepared by the present application realizes the safe transportation and functional exertion of mitochondria through the step-by-step design of "vector precise construction-function directional modification-content stable encapsulation-targeted efficient delivery".

[0023] Immune camouflage and long-term circulation: the hybrid vesicle composed of mesenchymal stem cell membrane and functionalized liposome inherits the "self" identification of mesenchymal stem cell membrane, which can effectively avoid the clearance of the body's immune system, and lays the foundation for long-term blood circulation and enrichment in lesion site.

[0024] Membrane anchoring synergistic effect of curcumin-thiol derivatives: The invention discards the traditional idea of simply dissolving curcumin in the preservation solution, and innovatively covalently anchors its thiol derivatives on the membrane structure of hybrid vesicles through click chemistry reaction. This design makes curcumin molecules become an inherent functional component of the vesicle membrane, and its action level is significantly improved: Membrane-located antioxidant barrier: Curcumin derivatives anchored on the membrane first contact and neutralize external free radicals during delivery, forming a dynamic active oxygen scavenging barrier on the periphery of the vesicle, providing "close-fitting" protection for the interior mitochondria, effectively resisting oxidative stress damage.

[0025] Membrane interaction promoter: The hydrophobic nature of curcumin molecules allows them to insert into the phospholipid bilayer of target cells, significantly reducing the membrane fusion energy barrier. This property plays a key role in promoting and bridging the close contact between the vesicle and the target cell membrane, and even the mitochondrial membrane, greatly improving the transmembrane delivery efficiency of the contents (i.e. functional mitochondria).

[0026] Precise active targeting: The modified TPP (triphenylphosphine) group on the functionalized liposome utilizes the mitochondrial membrane potential to drive the entire complex system to actively approach the mitochondrial network within the cell.

[0027] Precise delivery of functional mitochondria: Under the synergistic effect of TPP's precise navigation and curcumin derivatives' promotion of membrane interaction, the system ultimately delivers the internally encapsulated, optimally active mitochondria to the intended action site with high efficiency, achieving cell function repair.

[0028] Compared with the prior art, the core of the invention is to covalently fix curcumin-thiol derivatives on the vesicle membrane, which brings beneficial effects that are difficult to achieve by conventional methods such as "directly dissolving in the preservation solution". The reason why the invention discards the simple physical mixing scheme of directly dissolving curcumin in the mitochondrial preservation solution is that it has a significant negative impact on the activity and function of mitochondria: Curcumin, as a bioactive molecule, has been shown to exhibit beneficial antioxidant effects, but also concentration-dependent cytotoxicity. When directly dissolved in the closed preservation solution, its local concentration is extremely high, and it is in close contact with mitochondria for a long time. In this environment, curcumin molecules may no longer only play a protective role, but instead interfere with the integrity of the mitochondrial membrane, even induce the opening of the mitochondrial permeability transition pore (mPTP), leading to membrane potential collapse, swelling and even the release of apoptotic factors, thereby directly damaging the structure and function of the mitochondria to be delivered. In addition, curcumin is extremely unstable in a near-neutral or alkaline aqueous environment, and is prone to hydrolysis and oxidative degradation. Directly dissolved in the mitochondrial preservation solution (pH 7.4), it will accelerate its degradation process. These degradation products not only lose the original antioxidant activity, but may themselves have unknown chemical toxicity, causing unpredictable secondary damage to fragile mitochondria. Finally, high concentrations of free curcumin may non-specifically bind to proteins or enzymes on the mitochondrial membrane, competitively or non-competitively inhibiting the activity of respiratory chain complexes, disrupting oxidative phosphorylation, and directly weakening the energy production function of mitochondria. This is contrary to the fundamental purpose of transplanting functional mitochondria.

[0029] The present application covalently fixes curcumin in the form of a derivative on the membrane of the vesicle; the mitochondria are sealed in the inner water phase, and are physically isolated from the curcumin derivative on the membrane, thereby fundamentally avoiding the risk of toxicity caused by direct contact with high concentrations of curcumin. Covalent fixation creates a more stable "solid phase" reaction environment, significantly slowing down the degradation rate of curcumin, and ensuring its chemical stability and functional persistence throughout the delivery process. Its antioxidant function is "externalized" to the surface of the vesicle, used to scavenge external free radicals, rather than acting inside the mitochondria, thereby avoiding any potential interference with the core metabolic function of the mitochondria.

[0030] When the hybrid vesicle is successfully endocytosed by the target cell, the surface-fixed curcumin-thiol derivative is further activated in the intracellular environment. After the cell takes up the vesicle, an acidic endosome is usually formed. The hydrophobic nature of the curcumin derivative anchored on the membrane of the vesicle helps to disrupt the stability of the endosome membrane. It acts as a "membrane destabilizer", working synergistically with other components to promote the efficient escape of the vesicle from the endosome, avoiding its transport to the lysosome for degradation, and ensuring that the encapsulated active mitochondria can be successfully released into the cytoplasm.

[0031] The process of mitochondrial transplantation itself can trigger a transient intracellular oxidative stress. At this time, the curcumin derivative, released from the membrane of the vesicle or acting directly on its surface, as a powerful antioxidant, can immediately scavenge excess reactive oxygen species (ROS) in the cytoplasm, creating a low-oxidative-stress-friendly intracellular environment for the survival and functional integration of exogenous mitochondria.

[0032] The introduction of exogenous substances can activate inflammatory signaling pathways such as NF-κB in cells. The released curcumin derivatives can effectively inhibit the overactivation of these key inflammatory pathways, reduce the inflammatory response of cells, thereby protecting the host cells from damage, improving the acceptance of exogenous mitochondria by the host cells, and improving the overall cell survival rate.

[0033] The membrane anchoring strategy adopted in the present application is a design that converts potential toxicity risks into precise synergistic benefits, and is a key to ensure that the delivered mitochondria are "healthy, fresh and efficient".

[0034] In summary, the mitochondrial delivery system provided by the present application has significant beneficial effects: it achieves immune escape through mesenchymal stem cell membranes, prolongs in vivo circulation time, and achieves precise mitochondrial targeting through TPP modification. In particular, covalent anchoring of curcumin-thiol derivatives on the vesicle membrane forms a dynamic antioxidant barrier, providing whole-process protection for mitochondria, and significantly promoting membrane fusion and improving delivery efficiency. The concentration-dependent toxicity and functional interference that may be caused by directly dissolving curcumin in the preservation solution are avoided, and protection and delivery are achieved synergistically. Ultimately, the system can safely and efficiently deliver functional mitochondria to target cells, achieve functional repair, and exhibit great potential in the treatment of diseases related to mitochondrial dysfunction. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 、 Figure 2 、 Figure 3 、 Figure 4 、 Figure 5 、 Figure 6 are transmission electron microscope photos of mesenchymal stem cell mitochondria packaged in artificial vesicles at different magnifications. DETAILED DESCRIPTION

[0036] The technical solutions in the embodiments of the present application will be described below in a clear and complete manner. Obviously, the described embodiments are only some of the embodiments of the present application, not all. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0037] The experimental methods used in the detailed description are conventional methods unless otherwise specified, and the materials, reagents, etc. used are commercially available unless otherwise specified.

[0038] In the present application, unless otherwise specified, "%" represents mass percentage; the raw materials, reagents, etc. used are conventional commercially available products.

[0039] In the present application: DOPC refers to dioleoyl phosphatidylcholine; DSPE-PEG2000 refers to distearoylphosphatidylethanolamine-polyethylene glycol 2000; DSPE-PEG2000-TPP refers to distearoylphosphatidylethanolamine-polyethylene glycol 2000-triphenylphosphine; DSPE-PEG2000-Mal refers to distearoylphosphatidylethanolamine-polyethylene glycol 2000-maleimide; HEPES refers to 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid; DETA refers to ethylenediaminetetraacetic acid; DCC is N,N'-dicyclohexyl carbodiimide; DMAP is 4-dimethylaminopyridine; DMF refers to N,N-dimethylformamide; FBS refers to fetal bovine serum; succinate is sodium succinate; pyruvate is sodium pyruvate; malate is sodium malate; BSA refers to bovine serum albumin; protease inhibitor is Merck protease inhibitor cocktail.

[0040] Example 1 Synthesis of curcumin-thiol derivative The following steps are included: S1 curcumin activation: Take curcumin 3.0 mmol (1.11 g) and place it in a reaction bottle, add 15 mL of anhydrous DMF, and stir in the dark until it is completely dissolved; cool the reaction bottle to 0-5°C in an ice-salt bath; add 3.6 mmol of DCC and 0.3 mmol of DMAP in sequence; keep stirring at low temperature for 30 min; then stir at room temperature in the dark for 2 h; Use TCL detection (developing agent: dichloromethane:methanol=15:1), when a spot with Rf between 0.5-0.6 appears and the curcumin with Rf 0.3 becomes lighter, it means that the activation is successful.

[0041] S2 amidation reaction; (1) Preparation of 2-mercaptoethylamine free amine: Take 2-mercaptoethylamine hydrochloride 10 mmol) in a 50 mL flask; add 20 mL of anhydrous ether, and cool to 0°C in an ice bath; slowly add 2.1 mL of triethylamine; stir at room temperature for 2 hours, and filter to remove the triethylamine hydrochloride; dry the ether phase with anhydrous magnesium sulfate, filter, and rotary evaporate to obtain a colorless liquid, which is 2-mercaptoethylamine free amine.

[0042] (2) Nucleophilic attack of mercaptoethylamine Prepare 6.0 mmol 2-mercaptoethylamine free amine; dissolve in 5 mL anhydrous DMF; slowly drop into the reaction system through a constant pressure dropping funnel within 30 minutes; control the temperature not to exceed 30°C; After the drop is completed, remove the ice bath; stir the reaction at room temperature for 24 hours in the dark; Every 8 hours, sample TLC analysis, when the new product point Rf≈0.4-0.5, the raw material point gradually disappears, the reaction is complete.

[0043] S3 post-treatment and purification: (1) Reaction termination and preliminary treatment After the reaction is completed, pour the reaction liquid into 100 mL of ice ethyl ether to produce a large amount of white precipitate; filter through a Buchner funnel to collect the filtrate; Rotary evaporate the filtrate to remove most of the ethyl ether (water bath temperature ≤30°C); the remaining material is dissolved with 50 mL of ethyl acetate Wash with the following solutions in turn: 5% citric acid solution 3x30 mL Saturated NaHCO3 solution 3x30 mL Saturated NaCl solution 2x30 mL After washing, take the organic phase, dry overnight with anhydrous Na2SO4; filter to obtain the filtrate, and rotary evaporate to obtain orange-yellow crude product; (2) Column chromatography purification Column loading: Silica gel 200g, wet column loading (ethyl acetate) Column bed size: 3x30cm Gradient elution program: Stage: eluent ratio (ethyl acetate: petroleum ether): volume 1: (20:1): 500mL 2: (10:1): 500mL 3: (50:1): 800mL 4: (2:1): 500mL (3) Collection and identification TLC monitoring to collect the fractions (target product Rf=0.4-0.5, UV365nm coloration); combine the fractions containing the target product; rotary evaporate to remove the solvent; vacuum dry; obtain the product 1.18g (2.74mmol); yield 91.3%.

[0044] The product is sealed in a brown glass bottle under nitrogen and used as needed.

[0045] Example 2 Mesenchymal stem cell membrane extraction Including the following steps: S1 Cell culture and harvesting: (1 Cell source: Passage 3-5 human mesenchymal stem cells (hMSCs) from commercial channel and identified by flow cytometry (CD73, CD90, CD105 positive rate > 95%; CD34, CD45, HLA-DR negative rate < 2%).

[0046] Use mesenchymal stem cell special medium (such as MesenCult™) and add necessary supplements according to the instructions. Culture in a 15 cm culture dish at 37°C, 5% CO2 in a constant temperature incubator.

[0047] Harvest when the cell density reaches 80-90% confluence, ensure that the cells are in the best logarithmic growth phase, avoid excessive fusion leading to differentiation.

[0048] (2) After the culture is completed, the culture medium is aspirated, and the cells are gently washed twice with pre-cooled PBS; add 3 mL of preheated cell dissociation reagent and incubate at 37°C for 3-5 minutes.

[0049] Observe under a microscope, when about 80% of the cells are rounded and partially detached, gently tap the side wall of the culture dish to make the cells fall off. Add an equal volume of pre-cooled complete medium (containing 10% FBS) to terminate the digestion. Gently blow the cells with a pipette, collect the cell suspension, and centrifuge at 4°C, 300g for 5 minutes. Discard the supernatant, resuspend the cell pellet with pre-cooled PBS and wash twice to obtain the final cell suspension.

[0050] (3) Take 10 μL of cell suspension and count with a cell counting plate to obtain at least 5 x 10 7 cells for subsequent membrane extraction.

[0051] S2 cell membrane separation and purification (1) Hypotonic lysis: Resuspend the cell pellet in 10 mL of pre-cooled hypotonic lysis buffer (20 mM Tris-HCl, 1 mM EDTA, pH 7.5, and add 1 x protease inhibitor cocktail); incubate on ice for 30 minutes, gently vortex every 10 minutes; (2) Cell homogenate: Transfer the lysate to a pre-cooled Dounce homogenizer; gently grind until > 90% of the cells are broken and the nuclei are released; obtain the homogenate (3) Differential centrifugation purification: Transfer the homogenate to a centrifuge tube and centrifuge at 4°C, 800g for 10 minutes; collect the supernatant; repeat the homogenization of the pellet once and combine the supernatants; Centrifuge the supernatant at 4°C, 20,000g for 30 minutes; the pellet is the crude cell membrane fragment.

[0052] (4) Membrane fragment washing: Resuspend the membrane pellet with pre-chilled PBS, 4°C, 20,000g centrifuge for 30 minutes; repeat the washing for 2 times to make sure the cytoplasmic protein is removed (5) Membrane protein quantification: Resuspend the final membrane pellet with 1 mL PBS; take 10 μL to determine the protein concentration by BCA method; adjust to 2.0 mg / mL; obtain the desired mesenchymal stem cell membrane; aliquot and store at -80°C.

[0053] Example 3 Preparation of functionalized liposome and fixation of curcumin Set the surface density of maleimide in the hybrid vesicle σ = 5-15 / μm 2 ; According to the formula surface density σ = ([Mal]total × N a ) / (N ves × πd 2 ); Where [Mal]total is the total maleimide concentration in the hybrid vesicle suspension; N a is the Avogadro constant; N ves is the vesicle particle concentration; π is the constant π; d is the average diameter of the hybrid vesicle; Pre-set target hybrid vesicle: Average diameter d = 200 nm = 2 × 10 -7 m Target surface density σ = 10 / μm 2 (take the middle value) Each vesicle surface area A = π × d 2 = 3.1416 × (2 × 10 -7 ) 2 = 1.257 × 10 -13 m 2 = 0.1257 μm 2 The number of maleimides per vesicle = σ × A = 10 × 0.1257 ≈ 1.26 Therefore, according to the settlement results, the molar percentage of the components of the functionalized liposome is: DOPC, 65 mol% Cholesterol, 30 mol% DSPE-PEG2000, 4 mol% DSPE-PEG2000-TPP, 0.5 mol% DSPE-PEG2000-Mal, 0.5 mol%.

[0054] According to the formula, this embodiment includes the following steps: S1 Lipid weighing and thin film preparation Formulation calculation DOPC: 51.09 mg Cholesterol: 11.61 mg DSPE-PEG2000: 12.20 mg DSPE-PEG2000-TPP: 1.43 mg DSPE-PEG2000-Mal: 1.40 mg Using an analytical balance, weigh the above raw materials and sequentially dissolve them in chloroform / methanol (2:1, v / v) as the solvent: cholesterol first, then phospholipid, and finally PEGylated lipid; Perform rotary evaporation on the solution to form a film: water bath temperature: 30°C, rotation speed: 120 rpm, vacuum degree: <10 mbar; until a uniform transparent film is formed; vacuum dry the uniform transparent film overnight; S2 hydration and extrusion: Hydrate the uniform transparent film with hydration buffer (20 mM HEPES, 150 mM NaCl, pH 6.8) in a 50°C water bath for 1 hour with gentle shaking, then preheat the base device to 50°C, and extrude the 400 nm polycarbonate film 7 times; extrude the 200 nm polycarbonate film 13 times; maintain the extrusion pressure at 100-200 psi; S3 curcumin immobilization reaction: (1) According to DSPE-PEG2000-Mal moles = 0.182 mg / 2800 g / mol = 6.5 x 10 -8 mol; Assuming the recovery rate of liposomes after extrusion purification is 85%, the remaining DSPE-PEG2000-Mal mass in the system is approximately: 0.182 mg x 0.85 ≈ 0.155 mg The number of moles of DSPE-PEG2000-Mal in the system is approximately 5.54 x 10 -8 mol Calculate the amount of curcumin-thiol derivative required: Target reaction molar ratio: maleimide: curcumin-thiol = 1:1.2 Moles of curcumin-thiol derivative required: 5.54 x 10 -8 mol x 1.2 ≈ 6.65 x 10 -8 mol Mass of curcumin-thiol derivative required (MW = 468 g / mol): 6.65 x 10 -8 mol x 468 g / mol = 3.11 x 10 -5g = 0.0311 mg (2) Preparation of working solution Prepare primary stock solution: Accurately weigh 1.0 mg of curcumin-thiol derivative. Dissolve in 1.0 mL of anhydrous DMSO, vortex until completely dissolved. This primary stock solution concentration = 1.0 mg / mL.

[0055] Prepare secondary working solution: Accurately pipette 100 μL of primary stock solution (containing 0.1 mg of solute) using a micropipette.

[0056] Dilute 10-fold by adding 900 μL of anhydrous DMSO.

[0057] This secondary working solution concentration = 0.1 mg / mL.

[0058] Calculate and pipette the required volume: Required curcumin-thiol derivative mass = 0.0311 mg.

[0059] From the secondary working solution at a concentration of 0.1 mg / mL, pipette a volume of: 0.0311 mg / 0.1 mg / mL = 0.311 mL = 311 μL (3) Place the brown glass reaction vial containing 2 mL of functionalized liposomes on ice.

[0060] Bubble high purity nitrogen gas into the reaction vial for 10 minutes slowly through a needle inserted into the liquid level to remove dissolved oxygen.

[0061] While continuously bubbling nitrogen gas, add 311 μL of curcumin-thiol derivative / DMSO working solution dropwise into the liposome suspension using a micropipette, while gently shaking the vial to mix well.

[0062] (4) Quickly close the PTFE vial cap tightly to ensure a good seal. Place the reaction vial on a constant temperature shaker.

[0063] Reaction conditions: temperature: 25°C; rotation speed: 300 rpm; time: 12 hours; avoid light throughout.

[0064] (5) After 12 hours of reaction, remove the reaction vial.

[0065] Immediately purify using a size exclusion chromatography column (PD-10 Desalting Column) to terminate the reaction and remove unreacted curcumin derivative and DMSO.

[0066] First, equilibrate the column with 25 mL of HEPES buffer (pH 7.4).

[0067] The whole reaction solution (~2.3 mL) was carefully added to the column.

[0068] Elution was performed by adding 3.5 mL HEPES buffer (pH 7.4).

[0069] The first 2.5 mL of effluent was discarded, and the following 2.0 mL of light yellow opalescent eluate was collected. This was the purified curcumin-functionalized liposome (Cur-Lip).

[0070] Preparation of S4 hybrid vesicle membrane (1) The mesenchymal stem cell membrane and curcumin-functionalized liposome prepared in Example 2 were taken; the concentrations of the two were adjusted to a total lipid concentration of Cur-Lip of 4 mg / mL and a mesenchymal stem cell membrane protein concentration of 2 mg / mL with the membrane hybridization buffer; (2) The two were mixed at a ratio of lipid:membrane protein = 1:1 (w / w); gentle vortex mixing was performed to avoid the generation of bubbles; the components were pre-equilibrated on ice for 15 minutes.

[0071] (3) The SHM chip was installed on the microscope platform; the cooling circulation system was connected, and the temperature was set to 4°C; the chip channel was rinsed with 70% ethanol, then with sterile water, and finally with the membrane hybridization buffer.

[0072] Two 1 mL glass syringes were prepared: Syringe A: loaded with the Cur-Lip / membrane protein mixture (intermediate phase) Syringe B: loaded with HEPES buffer (sheath liquid) Bubbles in the syringes were excluded to ensure continuous liquid flow; the syringes were installed on the syringe pump and connected to the corresponding inlets of the chip.

[0073] Key process parameters: Total flow rate (TFR): 600 μL / min Flow rate ratio (FRR): intermediate phase: sheath liquid = 1:2 Intermediate phase flow rate = 600 x (1 / 3) = 200 μL / min Sheath liquid flow rate = 600 x (2 / 3) = 400 μL / min Temperature: 4°C Collection volume: all samples The syringe pump was started, and the hybridization process began; the flow state was monitored under a microscope to ensure the absence of bubbles and blockages.

[0074] (4) After hybridization, sucrose density gradient centrifugation was performed, and the gradient was prepared (from bottom to top): 2 mL of 60% sucrose (dissolved in HEPES buffer); 2 mL of 40% sucrose; 2 mL 20% sucrose; Add the hybrid sample; Centrifugation condition: speed: 100,000g; temperature: 4°C; time: 2 hours; acceleration: 5, deceleration: 3; After gradient centrifugation, take out the centrifuge tube and observe the band distribution; collect the opalescent band at the interface of 20%-40% with a pipette; the volume is about 2 mL; Dilute to 10 mL with HEPES buffer and ultracentrifuge (100,000g, 4°C, 30 minutes) Discard the supernatant and resuspend the precipitate with 1 mL of HEPES buffer; obtain the final hybrid vesicles, Example 4 Isolation of active mitochondria This example aims to isolate high-activity, intact mitochondria from human mesenchymal stem cells (hMSCs) for subsequent encapsulation and delivery applications.

[0075] The following steps are included: S1 Cell preparation and pretreatment: (1) Culture hMSCs to 80-90% confluence (T75 culture flask); discard the culture medium and wash the cells twice with pre-cooled PBS; add 2 mL of trypsin-EDTA and digest at 37°C for 3 minutes; add 4 mL of complete culture medium to terminate digestion; collect the cell suspension and centrifuge at 150g for 5 minutes; discard the supernatant and resuspend the cells with 10 mL of pre-cooled buffer A; Cell counting, target harvest amount 2 x 10 7 cells.

[0076] (2) In a low-temperature centrifuge, centrifuge at 150 x g for 5 minutes at 4°C. Carefully discard the supernatant without disturbing the cell precipitate. Gently resuspend the cell precipitate with 10 mL of pre-cooled mitochondria isolation buffer A.

[0077] Repeat the previous operation once; then resuspend the cell precipitate with 2 mL of pre-cooled mitochondria isolation buffer B, ready for the next homogenization operation.

[0078] (3) Transfer the cell suspension to a pre-cooled Dounce homogenizer; operate on ice and homogenize with a tight pestle: initial gentle grinding for 10 times; check the cell lysis efficiency (should be > 90%); Transfer the homogenate to a 15 mL centrifuge tube; First centrifugation: 600g, 4°C, 10 minutes, carefully collect the supernatant to avoid touching the precipitate; Second centrifugation: 11,000g, 4°C, 15 minutes, discard the supernatant and retain the precipitate; Resuspend the pellet gently with 1 mL of mitochondrial preservation solution; use a wide-bore pipette tip pre-chilled to avoid shear stress damage. Third centrifugation: 11,000 g, 4°C, 10 min, discard supernatant, keep mitochondrial pellet (4) Resuspend the mitochondrial pellet with appropriate volume of mitochondrial preservation solution, 10 7 mg / mL per 10 mg / mL per 10 mg / mL per 10 (5) Aliquot the mitochondrial suspension into pre-chilled 0.5 mL centrifuge tubes, quick-freeze; freeze condition: liquid nitrogen snap-freeze, -80°C storage (with 5% DMSO).

[0079] The formula of mitochondrial isolation buffer A is: 225 mM mannitol 75 mM sucrose 10 mM HEPES 1 mM EGTA pH 7.4 (adjusted with KOH), pre-chilled at 4°C The formula of mitochondrial isolation buffer B is: 225 mM mannitol 75 mM sucrose 10 mM HEPES 1 mM EGTA 0.1% BSA (fatty acid free) pH 7.4, pre-chilled at 4°C The formula of mitochondrial preservation solution is: 250 mM sucrose 50 mM mannitol 10 mM HEPES 5 mM KH2PO4 5 mM MgCl2 0.5 mM EDTA 5 mM sodium succinate 2 mM sodium pyruvate 1 mM sodium malate 0.1% (w / v) BSA 1x protease inhibitor cocktail.

[0080] Membrane potential was measured by flow cytometry (JC-1 staining) before freezing, Excitation / emission: 490 / 530 nm (monomer, green) Excitation / emission: 525 / 590 nm (J-aggregates, red) The result is the red / green fluorescence ratio > 3.0.

[0081] Mitochondrial encapsulation and final purification The purpose of this example is to encapsulate the biomimetic hybrid vesicles with active mitochondria by mild microfluidic technique to form the final product that can be used for delivery.

[0082] The following steps are included: S1 sample concentration adjustment: (1) Determine the lipid concentration of the biomimetic hybrid vesicles; determine the protein concentration of the mitochondria (BCA method); Mix according to the ratio of lipid: protein of mitochondria in hybrid vesicles = 8: 1 (w / w); adjust the total volume to 1.5 mL with mitochondrial preservation buffer (2) Vesicle labeling: take 0.8 mL of biomimetic hybrid vesicles (containing about 4 mg of lipid); place in a 1.5 mL brown centrifuge tube; add 1 μL of DiI ethanol solution (1 mg / mL); mix gently by vortex; Incubate on ice in the dark for 15 minutes, gently invert and mix every 5 minutes; Use 100 kDa molecular weight cut-off ultrafiltration tube, centrifuge at 4°C for 10 minutes at 4,000 g; repeat the washing 3 times to complete the vesicle labeling: Mitochondrial labeling: take 50 μL of freshly isolated mitochondria (containing about 0.5 mg of protein) and place in a 0.5 mL brown centrifuge tube; add 1 μL of MitoTracker Green (100 μM stock solution) to make the final concentration ≈ 200 nM; mix gently by blowing, incubate on ice in the dark for 15 minutes, gently mix by blowing every 5 minutes; use a small size exclusion column to remove free dye to complete the mitochondrial labeling; Mix the labeled hybrid vesicles and mitochondria, the total volume after mixing ≈ 2.05 mL, S2 microfluidic encapsulation: (1) Device setup: Use a new SHM chip, pre-cool at 4°C Syringe loading: Syringe A: vesicle-mitochondria mixture Syringe B: mitochondrial preservation buffer (containing mitochondria) (2) Encapsulation parameters Total flow rate (TFR): 800 μL / min Flow rate ratio (FRR): sample: buffer = 1:4 Sample flow rate = 800 x (1 / 5) = 160 μL / min Buffer flow rate = 800 x (4 / 5) = 640 μL / min Temperature: 4°C Processing times: single pass; (3) Start encapsulation process, collect export product; S3 Purification: (1) Density gradient centrifugation of export product; Preparation of discontinuous sucrose gradient: 60% sucrose: 2 mL 40% sucrose: 2 mL 20% sucrose: 2 mL Load: 2 mL of encapsulation product Centrifugation conditions: speed: 80,000 g; temperature: 4°C; time: 1.5 hours (2) Product collection and processing Collect the band at the interface of 30%-40% (successfully encapsulated mitochondria) After dilution with mitochondrial storage buffer, low-speed centrifugation speed: 500 g, time: 5 minutes; Collect the supernatant, filter sterilize with a 0.22 μm filter membrane, and adjust the final concentration to: mitochondrial protein: 4 mg / mL with mitochondrial storage buffer; freeze in liquid nitrogen.

[0083] Example 6 Related Verification (1) Transmission electron microscopy Take a transmission electron microscope photograph of the vesicles in Example 5 finally encapsulated with mesenchymal mitochondria, and the result is shown in FIG. 1- to Figure 6 As can be seen from the figure, the live mitochondria are located in the center of the image, and the bright, clear outline, and granular structure. Brightness indicates high density, and the internal structure (such as the ridge) is stained with a staining agent, and the clear outline indicates that the membrane structure is complete. The granular feeling comes from the internal biological macromolecules and ongoing metabolic activity. The dark shadow area around the mitochondria is a layer of membrane structure artificially synthesized by lipid molecules, which successfully encapsulates the mitochondria inside to form an independent delivery system.

[0084] (2) ATP content / synthesis rate detection ATP is the most direct energy currency of cells, and its level is a core indicator of mitochondrial function. It is measured based on the firefly luciferase-luciferin system. In the presence of luciferase and magnesium ions, ATP reacts with luciferin to generate oxy-luciferin and emit a light signal (~560 nm). The intensity of the light emission is proportional to the concentration of ATP in the sample.

[0085] Operation steps: S1 Sample preparation: Vesicle sample: Take a certain amount of vesicle suspension (containing 50 μg of protein), centrifuge at 12,000 g for 10 minutes at 4°C, discard the supernatant, and resuspend with pre-cooled PBS.

[0086] Cell sample: trypsinize and collect cells, count after PBS wash. Take the same number of cells for detection.

[0087] S2 sample lysis: According to the kit instructions, prepare the appropriate amount of ATP detection lysis solution.

[0088] Add 100 μL lysis solution to the precipitated complex or cells, vortex vigorously for 150 seconds, and lyse thoroughly.

[0089] After standing on ice for 5 minutes, centrifuge at 12,000g at 4°C for 5 minutes, and take the supernatant (i.e. ATP sample solution) and place it on ice.

[0090] S3 standard curve preparation: Take the ATP standard provided by the kit and perform a series of gradient dilutions with the lysis solution. Take 50 μL of each standard solution and add it to a white 96-well plate that is not transparent.

[0091] S4 detection reaction: Add 50 μL of the sample supernatant to the white 96-well plate. Prepare the ATP detection working solution. Add 100 μL of the working solution to each well.

[0092] Place the plate in the enzyme marker and mix quickly for 10 seconds, then immediately read the chemiluminescence value (RLU).

[0093] S5: data processing and expected results Calculation: according to the standard curve, convert the RLU value of the sample to ATP concentration (μM). Then normalize with the sample protein concentration measured by BCA method, and the final result is nmol ATP / mg protein.

[0094] The results are: Positive control (freshly isolated mitochondria): 252 nmol ATP / mg protein.

[0095] Experimental group (vesicles): 176 nmol ATP / mg protein.

[0096] Negative control (mitochondria treated with rotenone): 12 nmol ATP / mg protein.

[0097] (3) Quantitative analysis of mitochondrial membrane potential JC-1 is a "ratiometric" fluorescent probe.

[0098] Normal mitochondria (high membrane potential): JC-1 aggregates in the mitochondrial matrix, forming J-aggregates, emitting red fluorescence (590 nm). Functionally impaired mitochondria (low membrane potential): JC-1 exists in monomer form, emitting green fluorescence (529 nm).

[0099] The ratio of red / green fluorescence intensity is a direct indicator of membrane potential. A decrease in the ratio indicates depolarization of the membrane potential.

[0100] Operation steps: S1 Preparation of staining working solution: Dissolve JC-1 dye with ultrapure water according to the instructions, and prepare JC-1 staining buffer.

[0101] S2 Staining: Collect the vesicles and wash them once with PBS.

[0102] Resuspend the samples with JC-1 staining buffer and add an appropriate amount of JC-1 dye. Incubate in a 37°C cell incubator for 20-30 minutes in the dark.

[0103] S3 Washing: Wash the vesicles twice with pre-cooled JC-1 staining buffer, centrifuge at 4°C, 600g for 5 minutes to remove background fluorescence.

[0104] S4 Detection: Resuspend the samples with buffer and immediately detect them by flow cytometry.

[0105] Use a 488nm laser to excite, and use FL1 channel (green, 530 / 30nm) to detect JC-1 monomer and FL2 channel (red, 585 / 42nm) to detect JC-1 polymer, respectively.

[0106] Collect 10,000 valid events, analyze the average intensity of red and green fluorescence, and calculate the ratio.

[0107] S5: Data processing and expected results Calculation: Membrane potential = average fluorescence intensity ratio of FL2Red (590nm) / FL1Green (530nm).

[0108] The results are: Positive control (normal mitochondria): 7.24.

[0109] Experimental group (vesicles): 5.50 Negative control (samples treated with 10μM CCCP for 30 minutes): 0.057 (4) Quantitative analysis of intracellular reactive oxygen species level Experimental principle The CellROX probe itself has weak fluorescence and can penetrate the cell membrane. After entering the cell, it is oxidized by intracellular ROS to produce strong fluorescence, and the fluorescence intensity is proportional to the intracellular ROS level.

[0110] Operation steps: S1 Staining: Prepare cell samples. Set up positive control: treat cells with 1200 uM menadione for 1-2 hours to induce ROS production.

[0111] Add CellROX probe directly to cell culture medium to reach working concentration (2.5 uM); incubate in 37°C cell culture incubator for 30 minutes in the dark.

[0112] S2 Washing and Preparation: Gently wash vesicles twice with pre-warmed PBS; resuspend vesicles in PBS and transfer to flow tube or black 96-well plate.

[0113] S3 Flow Cytometry Detection: Immediately run on machine. CellROX Green: excitation / emission ~485 / 520 nm, use FL1 channel.

[0114] Collect 10,000 cells and analyze Mean Fluorescence Intensity (MFI) for channel of interest.

[0115] 4. Data Processing and Expected Results Calculation: calculate relative fluorescence intensity for each experimental group based on untreated normal cell group (set as 1).

[0116] Results are as follows: Positive control (menadione treatment): 5.2 Model group (MPP+-treated neuron cells): 3.4 Treatment group (model cells + vesicles): 2.1.

[0117] The above experimental results show that the composite vesicles encapsulating mesenchymal stem cells have good biological activity and functional efficacy.

[0118] Finally, it should be noted that: the above examples are used to illustrate the technical solutions of the present application, but not to limit it; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A mitochondrial delivery system, characterized in that, It consists of hybrid vesicles immobilized with curcumin-thiol derivatives and mitochondrial preservation fluid encapsulated by the hybrid vesicles; The hybrid vesicles are composed of a mesenchymal stem cell membrane and functionalized liposomes; and the amount of functionalized liposomes is greater than or equal to that of the mesenchymal stem cell membrane. The functionalized liposomes include at least the following components: DOPC, ≥50 mol% Cholesterol, ≥20 mol% DSPE-PEG2000, ≥2mol% DSPE-PEG2000-TPP, ≥0.2mol% DSPE-PEG2000-Mal, ≥0.5mol%.

2. The mitochondrial delivery system according to claim 1, characterized in that, The average diameter of the hybrid vesicles that do not encapsulate mitochondria is ≥200 nm.

3. The mitochondrial delivery system according to claim 1, characterized in that, The method for preparing the curcumin-thiol derivative is to activate the phenolic hydroxyl groups on curcumin, and then react them with an amino thiol compound to generate crude curcumin-thiol derivative; then, the crude curcumin-thiol derivative is obtained by purification.

4. The mitochondrial delivery system according to claim 3, characterized in that, The preparation method of the curcumin-thiol derivative includes the following steps: S1 Curcumin Activation: Dissolve curcumin in anhydrous dimethylformamide and cool in a water bath to ≤5℃; add N,N'-dicyclohexylcarbodiimide and 4-dimethylaminopyridine in sequence and stir to generate curcumin activation intermediate; S2 amidation reaction: The preparation method of curcumin-thiol derivative by freeing 2-mercaptoethylamine is to activate the phenolic hydroxyl groups on curcumin, and then react it with the free 2-mercaptoethylamine to generate crude curcumin-thiol derivative; then, after purification, the curcumin-thiol derivative is obtained. The amine is dissolved in anhydrous dimethylformamide and added dropwise to the reaction system of S1, and the reaction is stirred to obtain a crude curcumin-thiol derivative solution. S3 Post-processing and purification: The crude curcumin-thiol derivative solution was poured into ice-cold ether and filtered to collect the filtrate; the solution was removed by rotary evaporation, and the residue was dissolved in ethyl acetate, and then washed successively with citric acid solution, sodium bicarbonate solution, and sodium chloride solution; the organic phase was dried, filtered, and rotary evaporated to obtain the crude product; The crude product was purified by column chromatography to obtain curcumin-thiol derivatives.

5. The mitochondrial delivery system according to claim 1, characterized in that, The mesenchymal stem cell membrane of the hybrid vesicle is obtained by lysing, homogenizing, purifying by differential centrifugation, and washing cultured mesenchymal stem cells. The hybrid vesicle functionalized liposomes are obtained by mixing the components, rotary evaporation to form a membrane, hydration, nanomembrane extrusion, and purification. The hybrid vesicles are obtained by mixing mesenchymal stem cell membranes and functionalized liposomes in a buffer solution, hybridizing them using microfluidic technology, and then performing gradient centrifugation and resuspension.

6. The mitochondrial delivery system according to claim 1, characterized in that, The curcumin-thiol derivative is immobilized in the hybrid vesicles via a thiol-maleimide covalent bond; It includes the following steps: S1 pretreatment: Calculate the amount of each component to be added based on the target surface density of maleimide; weigh the required hybrid vesicles and curcumin-thiol derivative solution; S2 Click chemistry reaction: Dilute the hybrid vesicles to the target particle concentration with buffer solution, add the corresponding amount of curcumin-thiol derivative solution, and carry out the click chemistry reaction under light-protected conditions; S2 Click chemistry reaction: Dilute the hybrid vesicles to the target particle concentration with buffer solution, add the corresponding amount of curcumin-thiol derivative solution, and carry out the click chemistry reaction under light-protected and nitrogen-protected conditions; S3 Purification and Post-processing: After the reaction was completed, size exclusion chromatography was used to purify the product, remove unreacted curcumin derivatives and solvents, and collect the fraction containing hybrid vesicles with immobilized curcumin-thiol derivatives. In the preprocessing step S1: Surface density σ = ([Mal]total × N) a ) / (N ves ×πd 2 ); Where [Mal]total is the total maleimide concentration in the hybrid vesicle suspension; N a N is Avogadro's constant; ves denoted as vesicle particle concentration; π is pi; d is the average diameter of the hybrid vesicles. The surface density σ is 5-15 particles / μm. 2 .

7. The mitochondrial delivery system according to claim 1, characterized in that, Mitochondrial preservation fluid was encapsulated in hybrid vesicles immobilized with curcumin-thiol derivatives using a microfluidic encapsulation method. The mitochondrial preservation solution has a pH of 7.4 and contains at least the following components: Sucrose, 250mM; Mannitol, 50 mM; HEPES, 10mM; KH2PO4, 5mM; MgCl2, 5mM; EDTA, 0.5mM; Succinate, 5mM; Pyruvate, 2mM; Malate, 1 mM; BSA, 0.1% (w / v); Protease inhibitor, 1×; The 1× protease inhibitor refers to the corresponding 1x effective working concentration.

8. The mitochondrial delivery system according to claim 1, characterized in that, The mitochondria are immersed in a mitochondrial preservation solution, and the mitochondria are derived from mesenchymal stem cells.

9. A mitochondrial preparation, characterized in that, The mitochondria are encapsulated by the mitochondrial delivery system according to any one of claims 1-8.

10. Use of the mitochondrial preparation of claim 9 in the preparation of pharmaceuticals or health products related to mitochondrial dysfunction.

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