Extracellular vesicle-liposome hybrid nanoparticle and preparation method and application thereof

Abstract

The present application relates to the technical field of nano-preparation, in particular to an extracellular vesicle-liposome hybrid nanoparticle and a preparation method and application thereof. The extracellular vesicle-liposome hybrid nanoparticle provided by the present application significantly improves the ability of drugs to cross the blood-brain barrier and the target enrichment efficiency of ischemic lesions in the brain through active targeting modification; the specific response release of drugs is realized by using the high active oxygen microenvironment of the lesion, non-specific distribution of drugs is reduced, and the systemic toxic side effects are reduced; ultimately, a multi-target point synergistic treatment system capable of synchronously regulating neural cell apoptosis, glial cell inflammatory activation, brain microvascular endothelial cell ferroptosis and the structural integrity of the blood-brain barrier is constructed, the problem that the effect of traditional single target point intervention is limited is broken through from the root, and a more efficient, safe and precise new treatment strategy is provided for cerebral ischemia-reperfusion injury.

Description

Technical Field

[0001] This invention relates to the field of nanoparticle technology, and in particular to an extracellular vesicle-liposome hybrid nanoparticle, its preparation method, and its application. Background Technology

[0002] Ischemic stroke (IS) is a serious neurological disease, and cerebral ischemia-reperfusion injury (CIRI) is the core pathological mechanism of its pathogenesis and progression. When blood flow is restored after cerebral ischemia, it not only fails to fully restore neurological function but also leads to further aggravation of ischemic damage. Currently, the main clinical strategies for treating CIRI are intravenous thrombolysis and mechanical thrombectomy. However, both have strict time windows and can only restore blood flow, failing to effectively block secondary damage after reperfusion. Furthermore, the presence of the blood-brain barrier (BBB) ​​makes it difficult for most drugs to reach the brain lesion and exert their effects, resulting in poor clinical treatment outcomes.

[0003] The pathological process of CIRI (Chronic Inflammatory Reproductive Technology) is extremely complex, involving multiple interrelated pathological processes such as oxidative stress, neuroinflammation, apoptosis, ferroptosis, and blood-brain barrier disruption, forming a complex cascade of damage mechanisms. Among these, the excessive production of reactive oxygen species (ROS) is the core factor in initiating the cascade reaction. High ROS levels directly damage cellular macromolecules, activate microglia to release large amounts of pro-inflammatory factors, induce neuronal apoptosis and endothelial cell ferroptosis, ultimately leading to the disruption of the blood-brain barrier integrity and irreversible loss of neurological function. Single-target therapy is insufficient to comprehensively block this complex pathological cascade reaction; therefore, developing multi-target synergistic therapeutic strategies has significant clinical implications. Summary of the Invention

[0004] In view of this, the present invention provides an extracellular vesicle-liposome hybrid nanoparticle, its preparation method and application. The extracellular vesicle-liposome hybrid nanoparticle provided by the present invention blocks pathological cascade reactions through multi-target synergy, solving the problem that a single carrier is difficult to adapt to drugs with different physicochemical properties at the same time.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solution: In a first aspect, the present invention provides a method for preparing extracellular vesicle-liposome hybrid nanoparticles, comprising the following steps: (1) P53-siRNA was loaded into extracellular vesicles (EVs) to obtain extracellular vesicles of P53-siRNA (EVs-p53siRNA); P53-siRNA sequence: S: GGACAGCCAAGUCUGUUAUTT (SEQ ID NO: 1); AS: AUAACAGACUUGGCUGUCCTT (SEQ ID NO: 2); (2) 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-thioketal-polyethyleneglycol 2000-maleimide (DSPE-TK-PEG2000-Mal) was added to PBS buffer and dissolved by sonication; transferrin targeting peptide (HAIYPRH, C-T7 peptide) was added and reacted to obtain DSPE-TK-PEG2000-T7; (3) 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-Dioleoyl-3-trimethylammonium-propane (DOTAP), cholesterol, 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethyleneglycol) 2000 2000, DSPE-PEG2000), DSPE-TK-PEG2000-T7 and 7,8-dihydroxyflavone (7,8-DHF) were added to anhydrous ethanol, dissolved by sonication, and evaporated under reduced pressure to obtain a lipid film; the lipid film was hydrated, and the crude liposome suspension was treated with an ultrasonic water bath after hydration, and then extruded through a polycarbonate membrane to obtain 7,8-DHF-loaded liposomes (T7-liposomes). (4) Mix EVs-p53 siRNA with T7-liposomes and sonicate to freeze and thaw to obtain extracellular vesicle-liposome hybrid nanoparticles.

[0006] Extracellular vesicles (EVs) are naturally occurring nanoscale vesicles secreted by cells, possessing excellent biocompatibility, low immunogenicity, and natural blood-brain barrier penetration capabilities. EVs derived from human umbilical cord mesenchymal stem cells (hUC-MSCs) also naturally carry various neuroprotective factors; however, their endogenous brain-derived neurotrophic factor (BDNF) content is extremely low, resulting in very limited neuroprotective effects when used alone. 7,8-Dihydroxyflavone (7,8-DHF) is a small-molecule mimic of BDNF that specifically activates the tyrosine kinase receptor B (TrkB) signaling pathway, exerting neuroprotective effects comparable to BDNF, while also exhibiting significant antioxidant and anti-inflammatory activities. In addition, EVs themselves have limited anti-ferroptosis effects, while the P53 gene is a key regulator of ferroptosis. P53 small interfering RNA (P53-siRNA) can significantly enhance the anti-ferroptosis effect by silencing P53 expression and upregulating the expression of its downstream solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4), thus forming a multi-target synergistic therapy with 7,8-DHF.

[0007] Further, in step (1), the extracellular vesicles are obtained from P3-5 generation human umbilical cord mesenchymal stem cells through tangential flow ultrafiltration concentration, gradient centrifugation, and ultracentrifugation extraction. Human umbilical cord mesenchymal stem cells are easy to obtain, have few ethical constraints, and exhibit strong in vitro proliferative activity. Therefore, their preparation cost is low and they are easy to produce, making them more valuable for application compared to other cell sources. These cells have extremely low immunogenicity, resulting in highly safe and low-immunogenic extracellular vesicles. In the treatment of cerebral ischemia-reperfusion injury, these extracellular vesicles can efficiently penetrate the blood-brain barrier to target damaged brain tissue. They are rich in a large number of active substances, which can inhibit inflammatory responses, clear excess reactive oxygen species, reduce neuronal apoptosis, promote angiogenesis and neural tissue repair, improve the brain microenvironment, and have a better repair effect than other cell-derived extracellular vesicles.

[0008] Furthermore, tangential flow ultrafiltration concentration was performed using a 100 kDa membrane pack at a pressure <0.1 kPa to concentrate 500 mL of supernatant to 125 mL. The gradient centrifugation conditions were: 300 g for 10 min, 2000 g for 11 min, and 12000 g for 32 min, and the supernatant was collected. The ultracentrifugation conditions were: 120000 g for 75 min, the supernatant was discarded, and the precipitate was resuspended in sterile phosphate-buffered saline (PBS), and the ultracentrifugation was repeated once. After centrifugation, the supernatant was discarded, and the precipitate was resuspended in 200 μL of sterile PBS. The precipitate was then used after passing the characterization tests of nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and Western blotting.

[0009] Further, in step (1), P53-siRNA is loaded into EVs using electroporation.

[0010] The p53 gene is a key regulator of ferroptosis. Small interfering RNA (p53-siRNA) can significantly enhance the anti-ferroptosis effect by silencing p53 expression and upregulating the expression of its downstream SLC7A11 and glutathioneperoxidase 4 (GPX4), thus forming a multi-target synergistic therapy with 7,8-DHF.

[0011] Furthermore, in step (2), the molar ratio of DSPE-TK-PEG2000-Mal to C-T7 peptide is 1:1-1.1. The maleimide group on the DSPE-TK-PEG2000-Mal molecule can undergo specific covalent bonding with the thiol group in the C-T7 peptide.

[0012] Furthermore, in step (2), the reaction is carried out under nitrogen protection to avoid light. By isolating oxygen with nitrogen, the oxidative deactivation of thiol groups in the polypeptide molecule can be effectively avoided, while preventing the oxidative destruction of ketethiolated bonds and phospholipid structures. The light-protected operation can inhibit the hydrolytic deactivation of maleimide groups, reduce the photodegradation of photosensitive chemical bonds, ensure the smooth progress of the coupling reaction to the greatest extent, and improve the synthesis efficiency and structural stability of the product.

[0013] Furthermore, in step (2), the reaction temperature is 20-30 ℃ and the reaction time is 20-25 h.

[0014] Furthermore, in step (2), after the reaction is completed, dialysis and freeze-drying steps are also included; the dialysis operation is as follows: the reaction solution is transferred into a 3.5 kDa dialysis bag and dialyzed at 4 ℃ for 24 h.

[0015] Further, in step (3), the weight ratio of DOPC, DOTAP, Cholesterol, DSPE-PEG2000, DSPE-TK-PEG2000-T7 and 7,8-DHF is 26-28:8-10:4-6:1-3:2-4:4.

[0016] 7,8-DHF is a small molecule mimic of BDNF that can specifically activate the tyrosine kinase receptor B (TrkB) signaling pathway, exerting neuroprotective effects comparable to BDNF, while also possessing significant antioxidant and anti-inflammatory activities.

[0017] Furthermore, in step (3), the reduced pressure evaporation conditions are set as follows: reduced pressure evaporation in a rotary evaporator at 25-35 ℃, with a rotation speed of 50-70 rpm.

[0018] Further, in step (3), the hydration conditions are as follows: 7,8-DHF is dissolved in PBS to prepare a 2-3 mg / mL stock solution, and the lipid film is hydrated with this solution at 650-680 rpm for 50-70 min at room temperature.

[0019] Furthermore, in step (3), the specific ultrasound conditions are as follows: ultrasound power is set to 65-70W, ultrasound water bath treatment for 8-12 min, ultrasound for 8-12 s, and pause for 8-12 s. The intermittent ultrasound setting avoids the large thermal effect generated by continuous ultrasound, which would cause the lipid membrane to melt and break due to heat, and the loaded 7,8-DHF to degrade and become inactive due to heat. At the same time, it prevents the target modification bond from breaking, making the particle size finer and more uniform, and avoids excessive ultrasound causing breakage and a decrease in encapsulation rate.

[0020] Further, in step (3), the polycarbonate membranes are 400 nm and 200 nm polycarbonate membranes. Gradient extrusion is performed using polycarbonate membranes with pore sizes of 400 nm and 200 nm sequentially. First, the 400 nm membrane initially breaks down large-diameter vesicles, reducing the overall particle size of the system and decreasing the extrusion resistance of the subsequent small-diameter membrane, thus preventing damage to the liposome structure from strong extrusion. Then, the 200 nm membrane further sieves and regularizes the vesicles, precisely reducing the particle size and distribution width. Gradient extrusion can gradually control the vesicle morphology, efficiently obtaining liposomes with uniform particle size while maximally protecting the integrity of the phospholipid bilayer, reducing drug leakage and damage to the targeted modification structure, and ensuring the stability of the physicochemical properties and biological activity of the liposomes.

[0021] Furthermore, in step (4), the volume ratio of EVs-p53 siRNA to T7-liposomes is 2-4:1. When the ratio is too low, the proportion of extracellular vesicles is insufficient, making it difficult to fully integrate and fuse the siRNA delivery system, resulting in a low nucleic acid load and weakened gene silencing effect; when the ratio is too high, it is easy to cause excessive vesicle aggregation, leading to system aggregation and sedimentation, damaging the liposome brain-targeting structure, and increasing the risk of immune clearance in vivo.

[0022] Further, in step (4), the ultrasonic freeze-thaw treatment is as follows: the mixture of EVs-p53 siRNA and T7-liposomes is rapidly immersed in liquid nitrogen for 20-40 s to freeze, and then immediately placed in a water bath at 36-38℃ for rapid thawing for 20-40 s. This is one complete freeze-thaw cycle. This cycle is repeated 2-4 times. Then the mixture is placed at room temperature to recover for 8-12 min, and ultrasonic treatment is performed for 4-6 min.

[0023] In a second aspect, the present invention provides extracellular vesicle-liposome hybrid nanoparticles prepared by the preparation method described in the first aspect.

[0024] Thirdly, the present invention provides the application of the extracellular vesicle-liposome hybrid nanoparticles described in the second aspect in the preparation of drugs for treating cerebral ischemia-reperfusion injury.

[0025] The extracellular vesicle-liposome hybrid nanoparticles provided by this invention integrate the natural pharmacological activity and biological carrier function of EVs with the high encapsulation capacity of liposomes for hydrophobic drugs. The drug encapsulation rate of 7,8-DHF in the hybrid nanoparticles is 74.13%, and the drug loading is 5.93%, which solves the problem that a single carrier cannot simultaneously adapt to drugs with different physicochemical properties. Active brain targeting is achieved through C-T7 peptide modification, and responsive release in the lesion microenvironment is achieved by combining reactive oxygen species (ROS)-sensitive thionone bonds. The enrichment efficiency of intracranial lesions is greatly improved, and systemic toxic side effects are significantly reduced. At the same time, it intervenes in four core links: nerve cell apoptosis, glial cell inflammation, endothelial cell ferroptosis, and blood-brain barrier disruption. It stops the pathological process of CIRI from both primary and secondary damage dimensions, and the therapeutic effect is better than single drug administration.

[0026] Compared with the prior art, the present invention has achieved the following beneficial effects: The extracellular vesicle-liposome hybrid nanoparticles provided by this invention overcome the bottleneck that single nanocarriers cannot simultaneously and efficiently encapsulate hydrophobic small molecule drugs and hydrophilic nucleic acid drugs. Through active targeted modification, they significantly improve the drug's ability to cross the blood-brain barrier and the targeted enrichment efficiency of ischemic lesions in the brain. They utilize the highly reactive oxygen microenvironment of the lesion to achieve specific response release of the drug, reduce non-specific drug distribution and lower systemic toxic side effects. Ultimately, they construct a multi-target synergistic therapeutic system that can simultaneously regulate neuronal apoptosis, glial cell inflammatory activation, cerebral microvascular endothelial cell ferroptosis, and the structural integrity of the blood-brain barrier. This fundamentally overcomes the problem of limited efficacy of traditional single-target interventions, providing a more efficient, safe, and precise new treatment strategy for cerebral ischemia-reperfusion injury.

[0027] The materials used in the preparation method of this invention are all biocompatible natural or medical-grade materials, with no obvious organ toxicity or immunogenicity in vivo; the preparation process is simple and controllable, and easy to scale up, providing a new strategy and experimental basis for the clinical treatment of CIRI. Attached Figure Description

[0028] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0029] Figure 1 These are characterization diagrams of EVs; (A) is a particle size distribution diagram and a zeta potential diagram, (B) is a transmission electron micrograph of EVs, and (C) is a Western blot diagram of the marker protein of EVs. Figure 2 These are characterization diagrams of EVs-P53 siRNA; (A) is a particle size distribution diagram and Zeta potential diagram, (B) is a transmission electron micrograph, and (C) is a P53-siRNA content diagram. Figure 3 These are the characterization diagrams of the construction of DSPE-TK-PEG2000-T7; where (A) is the 1H-NMR result of DSPE-TK-PEG2000-Mal, (B) is the MALDI-TOF result of DSPE-TK-PEG2000-Mal, (C) is the 1H-NMR result of DSPE-TK-PEG2000-T7, and (D) is the MALDI-TOF result of DSPE-TK-PEG2000-T7. Figure 4 It is a fluorescence resonance curve; Figure 5These are particle size, potential, and PDI characterization diagrams of T7-liposomes and Hybrid NPs; where (A) is the particle size diagram of T7-liposomes, (B) is the potential diagram of T7-liposomes, (C) is the particle size diagram of Hybrid NPs, and (D) is the potential diagram of Hybrid NPs. Figure 6 These are transmission electron micrographs of T7-liposomes and Hybrid NPs; where (A) is a transmission electron micrograph of T7-liposomes and (B) is a transmission electron micrograph of Hybrid NPs. Figure 7 This is a graph showing the detection of EVs-p53 siRNA and surface proteins of Hybrid NPs; Figure 8 It is the standard curve diagram of 7,8-DHF; Figure 9 This is a release curve of Hybrid NPs under different conditions; Figure 10 This is an assessment diagram of HE staining in rat tissue; Figure 11 The diagrams show the distribution of different drugs in the major organs of rats. Among them, (A) shows the distribution of T7-liposomes and Hybrid NPs in the major organs, (B) shows the distribution of T7-liposomes and Hybrid NPs in the brain, (C) shows the relative fluorescence values ​​of T7-liposomes and Hybrid NPs in the brain, (D) shows the distribution of EVs-p53 siRNA and Hybrid NPs in the major organs, (E) shows the distribution of EVs-p53 siRNA and Hybrid NPs in the brain, and (F) shows the relative fluorescence values ​​of EVs-p53 siRNA and Hybrid NPs in the brain. Figure 12 These are scores of neurological deficits in rats after treatment with different drugs. Figure 13 The images show the cerebral infarction volume in MCAO rats after treatment with different drugs; (A) is a TTC staining image, and (B) is a quantitative statistical chart of the cerebral infarction area. Figure 14 This is a graph showing the effects of different drug treatments on neurogenesis in MCAO rats; Figure 15 This is a graph showing the effects of different drugs on the blood-brain barrier integrity in MCAO rats; Figure 16 These are staining images showing the effects of different drugs on the polarization of inflammatory cells in the brain tissue of MCAO rats. Figure 17This is a graph showing the effects of different drugs on the activation of inflammatory cells in MCAO rats. Detailed Implementation

[0030] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0031] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0032] Example 1 This embodiment provides a method for preparing extracellular vesicle-liposome hybrid nanoparticles, the specific preparation method is as follows: Step 1: Preparation of engineered extracellular vesicle (EVs)-p53 siRNA Human umbilical cord mesenchymal stem cells from generations 3-5 were collected. The culture supernatant was filtered through a 0.22 μm filter membrane for sterilization, followed by tangential flow ultrafiltration concentration, gradient centrifugation, and ultracentrifugation to extract extracellular vesicles from the cell supernatant. Extracellular vesicles in cell supernatant were extracted using tangential flow ultrafiltration concentration, gradient centrifugation, and ultracentrifugation. Tangential flow ultrafiltration concentration used a 100 kDa membrane pack at a pressure <0.1 kPa to concentrate 500 mL of supernatant to 125 mL. Gradient centrifugation was performed at 300 g for 10 min, 2000 g for 11 min, and 12000 g for 32 min, collecting the supernatant. Ultracentrifugation was performed at 120000 g for 75 min, discarding the supernatant, resuspending the precipitate in sterile PBS, and repeating ultracentrifugation once. After centrifugation, the supernatant was discarded, and the precipitate was resuspended in 200 μL of sterile PBS. The precipitate was then used after passing NTA, TEM, and Western blot characterization.

[0033] EVs-P53 siRNA was prepared by loading P53-siRNA into extracellular vesicles (EVs) using electroporation. The electroporation method was implemented as follows: The electroporation cuvette, centrifuge tube, and pipette tip were placed in a clean bench and irradiated with ultraviolet light for 30 min. 100 μL of purified extracellular vesicle suspension was added to a 1.5 mL RNase-free centrifuge tube. A 1-optical-density (1 OD) small interfering RNA (P53-siRNA) solution was added to the extracellular vesicle solution and gently mixed by pipetting. The mixture was slowly transferred to the electroporation cuvette, avoiding the formation of air bubbles. The electroporator was started, with the voltage set to 100-150 V, pulse duration 20-50 ms, pulse count 1, and interval 4 s. The electroporation cuvette was placed in the instrument slot, and the electroporation program was executed. After electroporation, the cuvette was placed at 37 ℃ for 20 min to allow for extracellular vesicle membrane repair.

[0034] The P53-siRNA sequences described in the experiment are: S: GGACAGCCAAGUCUGUUAUTT (SEQ ID NO: 1); AS: AUAACAGACUUGGCUGUCCTT (SEQ ID NO: 2).

[0035] Step 2: Synthesis of DSPE-TK-PEG2000-T7 functionalized lipid materials .

[0036] Add 5 mL of PBS reaction buffer to a 50 mL eggplant-shaped reaction flask. Accurately weigh 15 mg of DSPE-TK-PEG2000-Mal and add it to the buffer. Sonicate to dissolve for 10 min. Weigh the corresponding weight of C-T7 peptide (CHAIYPRH) at a molar ratio of 1:1 and add it to the reaction flask. React at room temperature in the dark under nitrogen protection for 24 h. Transfer the reaction solution to a 3.5 kDa dialysis bag and dialyze at 4 °C for 24 h to remove unreacted raw materials. Place the purified product DSPE-TK-PEG2000-T7 solution in a vacuum freeze dryer and freeze-dry until powdered. Collect the dried product and... 1 After successful coupling was verified by H-NMR and MALDI-TOF-MS, it was stored at -20℃ protected from light.

[0037] Step 3: Preparation of brain-targeting ROS-responsive cationic liposomes loaded with 7,8-DHF The weight ratio of each material was DOPC:DOTAP:Cholesterol:DSPE-PEG2000:DSPE-TK-PEG2000-T7:7,8-DHF = 27:9:5:2:3:4. 50 mg of each compound was accurately weighed, dissolved separately in anhydrous ethanol using ultrasound, and then added to a 50 mL round-bottom flask and mixed thoroughly. The mixture was then evaporated under reduced pressure (60 rpm / min) at 30°C using a rotary evaporator to form a uniform film. High-vacuum drying was continued for 2 h to remove residual solvent. 7,8-DHF was dissolved in PBS to prepare a 2 mg / mL stock solution. This solution was used to hydrate the lipid film at 660 rpm / min for 60 min at room temperature, ensuring complete detachment and full hydration. The specific conditions for ultrasound treatment were: 68W power, 10 min ultrasound in a water bath, 10 s ultrasound, 10 s pause, followed by extrusion through 400 nm and 200 nm polycarbonate membranes six times each, resulting in uniformly sized 7,8-DHF-loaded liposomes.

[0038] Step 4: Preparation of extracellular vesicle-liposome hybrid nanoparticles EVs-p53 siRNA and T7-liposomes were added to centrifuge tubes at a volume ratio of 3:1 and mixed thoroughly. The mixtures were then divided into three groups based on their mixing method: a direct mixing group of EVs-p53 siRNA and liposomes, a freeze-thaw mixing group of EVs-p53 siRNA and liposomes, and an ultrasonic freeze-thaw mixing group of EVs-p53 siRNA and liposomes.

[0039] The EVs-p53 siRNA and liposome direct mixing group was prepared by mixing EVs-p53 siRNA and liposomes evenly and then incubating at a constant temperature of 37℃ for 1 h to complete the mixing process. The EVs-p53 siRNA and liposome freeze-thaw mixing group was prepared by rapidly immersing the mixture in liquid nitrogen for 30 seconds, then immediately thawing it in a 37°C water bath for 30 seconds. This constituted one complete freeze-thaw cycle. This cycle was repeated 3 times to complete the mixing. The EVs-p53 siRNA and liposome ultrasonic freeze-thaw group first completed 3 freeze-thaw cycles according to the above freeze-thaw mixing group operation. Then, the mixture was placed at room temperature for 10 min to recover and then ultrasonically treated for 5 min. After treatment, the mixture was extruded through 400 nm and 200 nm polycarbonate membranes 3 times each to finally obtain extracellular vesicle-liposome hybrid nanoparticles. These nanoparticles were stored at 4℃ in the dark for later use.

[0040] Performance testing 1. Preparation method for fluorescence resonance detection of hybrid NPs NBD-PE and Rhodamine-PE with fluorescent probes were added to a round-bottom flask to synthesize NBD / Rho-liposomes labeled with both fluorescence.

[0041] The above three methods were used to treat the mixture of extracellular vesicles and liposomes. The fluorescence spectrophotometer was started, and 100 μL of the mixture after each treatment was added to a 96-well black microplate. The plate was placed in the instrument's detection chamber, and the fluorescence emission spectra of individual liposomes and the three treatment groups were detected in sequence. The characteristic peaks were recorded and the spectral curves were plotted.

[0042] 2. Safety evaluation Establishment of a rat model of middle cerebral artery occlusion (MCAO): Rats were anesthetized and fixed supine on an operating table. Hair was shaved from the midline of the rat's neck. A 1.5-2 cm longitudinal incision was made in the midline of the rat's neck. The external carotid artery and its branches were ligated with 4-0 sutures. The proximal end of the common carotid artery was then ligated, and the distal end of the internal carotid artery was clamped with an arterial clamp. A slipknot was tied in the middle of the common carotid artery. Approximately 0.5 cm below the bifurcation of the common carotid artery, a small oblique incision was made with ophthalmic scissors. Using ophthalmic forceps, the tip of the suture thrombus was held and slowly inserted through the incision in the common carotid artery. Once resistance was felt at the bifurcation point, the insertion was stopped, the slipknot was tightened, and the thrombus was gently secured. The neck skin incision was sutured with 2-0 sutures. Occlusion of the middle cerebral artery was completed after 2 hours of ischemia. Two hours after ischemia, the skin incision was opened to expose the suture plugs. All the suture plugs were slowly removed to restore blood supply to the middle cerebral artery and achieve reperfusion. The skin incision was then sutured again to ensure no bleeding.

[0043] Biosafety evaluation: To test the biosafety of the drug in animals, rats were first modeled by middle artery occlusion and reperfusion and then treated with the drug. Five days after administration via tail vein injection, heart, liver, spleen, lung, and kidney tissues of rats were collected and analyzed by HE staining to observe for any obvious pathological damage, inflammatory cell infiltration, tissue necrosis, or other abnormalities.

[0044] 3. Targeted evaluation T7-liposomes were labeled with Rhodamine B dye, and EVs-p53 siRNA was labeled with 1,1'-Dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt (DID) dye. After rats were modeled and administered the drugs, brain tissue and other organs were dissected 24 h later, and images were acquired using a small animal in vivo imaging system.

[0045] 4. In vivo pharmacodynamic evaluation (1) Rats' neurological deficit score After establishing the model, the drug was administered, and neurological behavioral scores were assessed according to the Longa scoring system 5 days later. 0 points: No neurological deficits, rats move normally, and limbs extend symmetrically; 1 point: Mild defect, flexion and adduction of the paralyzed forelimb in rat; 2 points: Moderate defect, the rat turns to the left when walking and has limited limb movement; 3 points: Severe defect; the rat leans to the left when walking and is unable to maintain its balance on its own. 4 points: Profound defect, rat with impaired consciousness, no spontaneous activity, only limb reflexes; 5 points: The rat died.

[0046] (2) TTC staining of brain tissue and measurement of infarct volume After drug administration to the affected animals, the animals were sacrificed 5 days later, and brain tissue was quickly collected and frozen at -20 ℃ for 30 min. Five 2 mm thick brain slices were cut from each rat brain tissue. The slices were immersed in 2% 2,3,5-triphenyltetrazolium chloride (TTC) staining solution and incubated at 37 ℃ in the dark for 20 min, gently shaking every 5 min to ensure uniform staining. After staining, the slices were fixed with 4% paraformaldehyde for 24 h, then placed on black cardstock and photographed. Results: Normal brain tissue stained red with TTC, while ischemic infarcted brain tissue appeared white due to cell necrosis and enzyme inactivation, preventing TTC reduction. The infarct area was calculated using ImageJ software; the infarct volume percentage was calculated as infarct volume / cerebral hemisphere volume × 100%.

[0047] (3) Detection of brain tissue cell apoptosis by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Five days after modeling and drug administration, rats were sacrificed and their brains were collected and fixed for 24 hours. The sections were immersed three times in xylene solution for 10 minutes each time, followed by three times in ethanol for 5 minutes each time, and finally washed with distilled water; incubated with proteinase K working solution for 20 minutes. After washing with PBS, they were incubated with permeabilization solution for 20 minutes. 5% bovine serum albumin (BSA) blocking solution was added, and the sections were incubated at 37°C for 1 hour. The blocking solution was discarded, and no washing was performed. Neuronal class III β-tubulin (TUJ1) primary antibody was diluted with 5% BSA and added to the sections. The sections were incubated overnight at 4°C in the dark, and washed three times with PBS for 5 minutes each time to remove unbound primary antibody. Fluorescently labeled secondary antibody was added, and the sections were incubated at 37°C in the dark for 1 hour. The sections were washed three times with PBS for 5 minutes each time, and the process was performed in the dark to prevent fluorescence quenching. Following the TUNEL kit instructions, add the kit reaction solution and incubate at 37°C in the dark for 30 min. Wash three times with PBS, 5 min each time, to specifically label apoptotic cells. Add 4′,6-diamidino-2-phenylindole (DAPI) staining solution and incubate at room temperature in the dark for 5 min. Wash three times with PBS, 5 min each time, to stain the nuclei and locate them. Add anti-fluorescence quenching mounting medium, cover with a coverslip, avoiding air bubbles, and observe under a laser confocal microscope.

[0048] (4) Zonula occludens protein 1 (ZO-1) staining to detect the effect of drugs on the integrity of the blood-brain barrier. Brain tissue from rats after drug administration was collected for modeling, fixed with 4% paraformaldehyde, routinely dehydrated, embedded in paraffin, and prepared into coronal sections. After dewaxing and rehydration, antigen retrieval was performed, followed by blocking with 5% BSA, incubation with ZO-1 primary antibody at 4 ℃ overnight, incubation with fluorescent secondary antibody the next day, nuclear staining with DAPI, and mounting with anti-fluorescence quenching. Images were acquired under a laser confocal microscope to assess the effect of the drug on the integrity of the tight junction of the blood-brain barrier.

[0049] (5) Inducible nitric oxide synthase (iNOS) / differentiation antigen 206 (mannose receptor, CD206) staining to detect the effect of drugs on inflammatory cells in brain tissue. Brain tissue from rats after drug administration was collected for modeling, fixed with 4% paraformaldehyde, routinely dehydrated, embedded in paraffin, and prepared into coronal sections. After dewaxing, rehydration, and antigen retrieval, the sections were blocked with 5% BSA, and incubated overnight at 4 ℃ with iNOS and CD206 primary antibody. The next day, the sections were incubated with the corresponding fluorescent secondary antibody, stained with DAPI, and mounted with anti-fluorescence quenching. Images were acquired using a laser confocal microscope to evaluate the regulatory effect of the drug on inflammatory cells in brain tissue.

[0050] (6) Detection of the effect of drugs on inflammatory cells in brain tissue by staining for ionized calcium-binding adapter molecule 1 (Iba1) / glial fibrillary acidic protein (GFAP). Brain tissue from rats after drug administration was collected for modeling, fixed with 4% paraformaldehyde, routinely dehydrated, embedded in paraffin, and prepared into coronal sections. After dewaxing, rehydration, and antigen retrieval, the sections were blocked with 5% BSA, and incubated overnight at 4 ℃ with IBA1 and GFAP primary antibodies. The next day, the sections were incubated with the corresponding fluorescent secondary antibody, stained with DAPI, and mounted with anti-fluorescence quenching. Images were acquired using a laser confocal microscope to evaluate the regulatory effect of the drug on inflammation-related cells in brain tissue.

[0051] Results and Discussion Figure 1 These are characterization diagrams of EVs; (A) is a particle size distribution diagram and a zeta potential diagram, (B) is a transmission electron micrograph of EVs, and (C) is a Western blot diagram of the marker protein of EVs.

[0052] Figure 1 Results (A) show that the particle size of the EVs is 106.6 ± 65.0 nm and the potential is -9.41 ± 0.56 mV, which meets the requirements. Figure 1 The (B) TEM results show that the EVs have a cup-shaped circular structure under the transmission electron microscope, and the double membrane structure can be observed. The vesicle boundaries are clear, which meets the requirements. Figure 1(C) The results of characteristic marker protein detection of the extracted EVs showed that clear and specific target bands appeared at the theoretical molecular weight positions of differentiation antigen 63 (CD63), differentiation antigen 81 (CD81), and tumor susceptibility gene 101 (TSG101), indicating that the extracted samples expressed characteristic proteins of EVs. However, no specific bands were detected for calnexin and Golgi matrix protein 130 (GM130) in the EV samples, suggesting that the extracted EV samples had good purity. In summary, the EVs extracted in this experiment met the characteristic protein identification criteria for extracellular vesicles, confirming successful EV extraction and that the sample purity met the requirements for subsequent in vivo and in vitro functional experiments.

[0053] Figure 2 These are characterization diagrams of EVs-P53 siRNA; (A) is a particle size distribution diagram and Zeta potential diagram, (B) is a transmission electron microscopy image, and (C) is a P53-siRNA content diagram.

[0054] Figure 2 Results (A) showed that the particle size of EVs-p53 siRNA was 124.2 ± 40.1 nm, and the Zeta was -9.93 ± 0.61 mV. The particle size and potential were not significantly different from those of EVs, and no obvious changes were observed. Figure 2 As observed under TEM in (B), EVs-p53 siRNA still maintains the typical morphological characteristics of EVs, presenting as a round or cup-shaped vesicle structure with intact membrane, no obvious rupture or morphological abnormalities, consistent with the morphological structure of EVs. Figure 2 The qPCR results in (C) showed that, compared with the EVs group, the EVs-p53 siRNA group showed significantly high expression of P53-siRNA, while no specific signal of P53-siRNA was detected in the EVs group.

[0055] The above results confirm that p53-siRNA can be successfully loaded onto EVs using electroporation, and this loading method does not significantly affect the core physicochemical properties of EVs. EVs-p53 siRNA still maintains the original particle size, potential characteristics and complete morphological structure of EVs.

[0056] Figure 3These are the characterization diagrams of DSPE-TK-PEG2000-T7; where (A) is the 1H-NMR result of DSPE-TK-PEG2000-Mal, (B) is the MALDI-TOF result of DSPE-TK-PEG2000-Mal, (C) is the 1H-NMR result of DSPE-TK-PEG2000-T7, and (D) is the MALDI-TOF result of DSPE-TK-PEG2000-T7.

[0057] Figure 3 Results (A) show that the DSPE-TK-PEG2000-Mal active pharmaceutical ingredient exhibits a characteristic singlet of maleimide group at a shift of 6.7 ppm. Figure 3 Results (B) show that the molecular weight of DSPE-TK-PEG2000-Mal, as detected by MALDI-TOF-MS, is around 3399.8. Figure 3 The results in (C) show that the characteristic singlet of the product DSPE-TK-PEG2000-T7 after the reaction of DSPE-TK-PEG2000-Mal with C-T7 peptide completely disappeared at a shift of 6.7 ppm. Figure 3 The results (D) show that the molecular weight of DSPE-TK-PEG2000-T7 detected by MALDI-TOF-MS is around 4172.4. The actual molecular weight of the reaction product is in high agreement with the calculated theoretical molecular weight of the coupling product, which is approximately 4172, with no significant deviation. Furthermore, the target peak in the mass spectrum is single and without significant impurity peaks, indicating high purity of the reaction product and no obvious unreacted raw materials or byproduct residues. In summary, combined with... 1 Qualitative analysis of functional groups by H-NMR and quantitative verification of molecular weight by MALDI-TOF-MS confirm that the coupling reaction between DSPE-TK-PEG2000-Mal and C-T7 peptide was successfully carried out, the target conjugate was successfully synthesized, and the product structure meets the theoretical design requirements.

[0058] Figure 4 It is a fluorescence resonance curve.

[0059] To investigate the effects of different treatment methods on the fusion efficiency of EVs and dual-fluorescently labeled liposomes (NBD / Rho-liposomes), this experiment first prepared NBD / Rho-liposomes dual-fluorescently labeled with nitrobenzoxadiazole-labeled phosphatidylethanolamine (NBD-PE) and rhodamine-labeled phosphatidylethanolamine (Rhodamine-PE). Then, different treatment groups were set up to treat the EVs-p53 siRNA and NBD / Rho-liposomes mixture system. The FRET signal changes of each group were detected by fluorescence spectrophotometer to evaluate the fusion effect of different methods. The following groups were set up: a liposome control group (NBD / Rho-lips), an EVs-p53 siRNA and liposome direct mixing group (NBD / Rho-lips+EVs directmixing), an EVs-p53 siRNA and liposome freeze-thaw group (NBD / Rho-lips+EVs freeze-thaw), and an EVs-p53 siRNA and liposome ultrasonic freeze-thaw group (NBD / Rho-lips+EVs ultrasonic freeze-thaw).

[0060] like Figure 4 As shown, no significant FRET signal changes were observed in the NBD / Rho-lips group and the NBD / Rho-lips+EVs direct mixing group. Compared with the NBD / Rho-lips + EVs direct mixing group, the NBD / Rho-lips+EVs freeze-thaw group exhibited significant FRET, with a marked change in fluorescence emission spectral characteristics. The NBD / Rho-lips+EVs ultrasonic freeze-thaw group showed the most significant FRET phenomenon, with a significantly higher fluorescence value at the characteristic emission wavelength of the energy donor NBD (535 nm) compared to the direct mixing and freeze-thaw groups, and a significantly lower fluorescence value at the characteristic emission wavelength of the energy acceptor Rhodamine B (635 nm) compared to the other two groups. The spectral curves exhibited typical FRET signal change characteristics. These results indicate that the fusion efficiency of EVs and NBD / Rho-liposomes is related to the treatment method. Freeze-thaw treatment can significantly improve the fusion effect, while ultrasonic freeze-thaw treatment can further improve the fusion efficiency, resulting in more complete energy transfer between the FRET donor and acceptor.

[0061] Figure 5These are particle size, potential, and PDI characterization diagrams of T7-liposomes and Hybrid NPs; where (A) is the particle size diagram of T7-liposomes, (B) is the potential diagram of T7-liposomes, (C) is the particle size diagram of Hybrid NPs, and (D) is the potential diagram of Hybrid NPs. Figure 5 Results (A) show that the average particle size of T7-liposomes is 166.9 nm and the polydispersity index (PDI) is 0.149. Figure 5 Results (B) show that the T7-liposomes zeta potential is 6.36 mV; Figure 5 The (C) results show that the average particle size of the Hybrid NPs is 191.3 nm and the PDI is 0.145; Figure 5 The (D) results show that the Zeta potential of the Hybrid NPs is 2.41 mV. The PDI of both nanomaterials is less than 0.3, indicating that they have uniform particle size distribution, good dispersibility, and no obvious agglomeration.

[0062] Figure 6 These are transmission electron micrographs of T7-liposomes and Hybrid NPs; where (A) is a transmission electron micrograph of T7-liposomes and (B) is a transmission electron micrograph of Hybrid NPs.

[0063] The morphology of the prepared liposomes and hybrid NPs was observed using transmission electron microscopy. The results are as follows: Figure 6 As shown. Figure 6 The results in (A) showed that T7-liposomes had a uniform morphology, were irregularly round, had a rough surface and rough edges, were well dispersed, and had no obvious aggregation. Figure 6 Results (B) show that the Hybrid NPs exhibit regular circular or near-circular vesicle structures under transmission electron microscopy. Compared with single liposomes, the membrane structure of the fused particles is more compact and has good morphological uniformity. No obvious aggregation was observed, indicating that the extracellular vesicles and liposomes have successfully undergone membrane fusion to form a structurally stable and morphologically regular composite nanocarrier.

[0064] Figure 7 This is a graph showing the detection of EVs-p53 siRNA and surface proteins of Hybrid NPs.

[0065] The results are as follows Figure 7As shown, no CD63, CD81, or TSG101 expression bands were detected in the mesenchymal stem cell (MSC) group, while these bands were detected in the EVs, EVs-p53 siRNA, and Hybrid NPs groups. Calnexin and GM130 expression were detected in the MSC group, but not in the EVs, EVs-p53 siRNA, and Hybrid NPs groups. These results indicate that the expression of extracellular vesicle surface marker proteins in EVs-p53 siRNA treated with p53-siRNA remained unchanged; while Hybrid NPs prepared by fusion of EVs-p53 siRNA with liposomes still fully retained the surface marker protein characteristics of EVs, completely consistent with the surface marker protein expression results of EVs and EVs-p53 siRNA. In summary, neither the electroporation process of p53-siRNA nor the process of fusing EVs with liposomes to prepare Hybrid NPs after electroporation significantly affected the expression of extracellular vesicle surface marker proteins, and Hybrid NPs could retain the protein expression characteristics of EVs.

[0066] Figure 8 It is the standard curve of 7,8-DHF.

[0067] A standard curve for 7,8-DHF was established using ultraviolet spectrophotometry. A drug quantification method was constructed to accurately quantify the concentration of 7,8-DHF. The free drug and drug carrier were then separated by dextran gel column centrifugation, and the drug concentration was determined by ultraviolet spectrophotometry. The encapsulation efficiency and drug loading of the two drug loading systems were calculated and evaluated.

[0068] The absorbance of various dilutions of 7,8-DHF was prepared and measured. A linear regression analysis was performed with the 7,8-DHF drug concentration on the x-axis and the corresponding absorbance values ​​on the y-axis to establish a UV-Vis quantitative standard curve for 7,8-DHF. The results are as follows: Figure 8As shown, 7,8-DHF exhibits good linearity within the concentration range of 0-16 μg / mL, indicating that this method can be used for subsequent quantitative detection of 7,8-DHF concentration. Free 7,8-DHF was effectively separated from T7-liposomes and hybrid NPs using dextran gel column centrifugation. The separated drug-loaded samples were demulsified, and the absorbance values ​​were measured using ultraviolet spectrophotometry. The actual drug concentration in the samples was calculated using the 7,8-DHF standard curve. Relevant indicators for the two drug loading systems were calculated based on the encapsulation efficiency and drug loading formulas. The results showed that the drug encapsulation efficiency of 7,8-DHF in liposomes was 81.10%, and the drug loading was 6.49%; the drug encapsulation efficiency of 7,8-DHF in hybrid nanoparticles was 74.13%, and the drug loading was 5.93%. These results indicate that both drug loading systems exhibit good drug encapsulation and loading capabilities, meeting the experimental requirements for subsequent in vitro and in vivo delivery and efficacy studies of 7,8-DHF.

[0069] Figure 9 This is a release curve of Hybrid NPs under different conditions.

[0070] In this experiment, ROS-sensitive TK groups were introduced during the construction of liposomes. These groups can break down under oxidative stress, thereby achieving responsive drug release. To verify the ROS-responsive drug release characteristics of the synthesized hybrid nanoparticles (H2O2), H2O2 concentration gradients of 10 μM, 100 μM, 1 mM, and 10 mM were set up to investigate the in vitro release behavior of 7,8-DHF from hybrid nanoparticles (Hybrid NPs) at different ROS concentrations, clarifying the correlation between their drug release characteristics and H2O2 concentration.

[0071] Experimental results are as follows Figure 9 As shown, 7,8-DHF loaded on Hybrid NPs exhibited significant ROS-responsive release characteristics in H2O2 media at different concentrations. Both the drug release rate and cumulative release rate showed a continuous upward trend with increasing incubation time. Furthermore, this release behavior exhibited a significant concentration dependence; with increasing H2O2 concentration, the release rate of 7,8-DHF significantly accelerated, and the cumulative release rate also significantly increased for the same incubation time. Specifically, drug release was relatively slow at 10 μM and 100 μM H2O2 concentrations, while the release rate significantly increased at 1 mM and 10 mM H2O2 concentrations, and the cumulative release rate was significantly higher than the low-concentration group, demonstrating a response-based release pattern matched to ROS concentration. These results confirm that Hybrid NPs can achieve oxidative stress-dependent release of 7,8-DHF through the specific response of the TK group on the liposomes to ROS, and that the release behavior is positively correlated with ROS concentration.

[0072] Figure 10 This is an assessment diagram of HE staining in rat tissue.

[0073] To evaluate the systemic safety of rats after intervention with MCAO, 7,8-DHF, T7-liposomes, Hybrid NPs, and EVs-p53 siRNA, this study selected five major organs—heart, liver, spleen, lung, and kidney—and used hematoxylin-eosin staining (HE staining) to observe morphological changes in the tissues.

[0074] like Figure 10 As shown, the HE-stained sections of heart, liver, spleen, lung, and kidney tissues from rats in the sham operation group (Sham group) exhibited intact structures, regular cell arrangement, and no pathological changes such as inflammatory infiltration, edema, or necrosis. Compared with the Sham group, there were no significant differences in cell morphology, arrangement, and histological characteristics in HE-stained sections of heart, liver, spleen, lung, and kidney tissues from rats in the MCAO model group, 7,8-DHF group, T7-liposomes group, Hybrid NPs group, and EVs-p53 siRNA group. No pathological damage such as inflammatory cell infiltration, hepatocyte hydropic degeneration, renal tubular necrosis, or alveolar structural destruction was observed, preliminarily confirming that the drug and formulation combination has good systemic safety at the experimental dosage and cycle.

[0075] Figure 11 These are diagrams showing the distribution of different drugs in major organs of rats. Specifically, (A) shows the distribution of T7-liposomes and Hybrid NPs in major organs, (B) shows the distribution of T7-liposomes and Hybrid NPs in the brain, (C) shows the relative fluorescence values ​​of T7-liposomes and Hybrid NPs in the brain, (D) shows the distribution of EVs-p53 siRNA and Hybrid NPs in major organs, (E) shows the distribution of EVs-p53 siRNA and Hybrid NPs in the brain, and (F) shows the relative fluorescence values ​​of EVs-p53 siRNA and Hybrid NPs in the brain.

[0076] To compare the tissue distribution characteristics of different drug formulations in MCAO model rats and clarify the drug's blood-brain barrier penetration ability and brain targeting accumulation effect, this experiment collected fluorescence signals from rat heart, liver, spleen, lung, kidney, and brain tissues after tail vein injection. The brain distribution pattern of the drug was qualitatively and quantitatively analyzed. The experimental groups were: T7-liposomes group (Rhodamine B-labeled T7-liposomes), EVs-p53 siRNA group (DID-labeled EVs), and HybridNPs group (DID-labeled EVs, Rhodamine B-labeled T7-liposomes).

[0077] (1) Results of fluorescence signals in isolated organs as follows Figure 11 As shown in Figure (A), by detecting Rhodamine B fluorescence, significant Rhodamine B fluorescence signals were detected in the liver, spleen, and lung tissues of both the T7-liposomes and Hybrid NPs groups, indicating that both formulations, after tail vein injection, can be distributed in the peripheral metabolic and clearance organs of rats. The results differ from those in peripheral organ distribution, as shown in Figure (A). Figure 11 As shown in Figures (B) and (C), there were significant differences in Rhodamine B fluorescence signals in the brain tissues of the two groups of rats: high-intensity fluorescence signals were detected in the brains of rats in the Hybrid NPs group, with a significantly higher degree of fluorescence enrichment than in the T7-liposomes group; while only weak fluorescence signals were observed in the brains of the T7-liposomes group, indicating extremely low brain-targeted delivery efficiency. These results confirm that Hybrid NPs can penetrate the blood-brain barrier more efficiently and achieve significant enrichment at sites of brain ischemia-reperfusion injury.

[0078] (2) Results of fluorescence signals in isolated organs as follows Figure 11 As shown in Figure (D), both the EVs-p53 siRNA group and the HybridNPs group showed significant DID fluorescence signals in the liver, spleen, and lung tissues, indicating that both formulations, after tail vein injection, could be distributed in the peripheral metabolic and clearance organs of rats. Figure 11 As shown in Figures (E) and (F), high-intensity and significantly enriched DID fluorescence signals were detected in the brains of rats in the Hybrid NPs group, with fluorescence brightness and signal range much higher than those in the EVs-p53 siRNA group. These results confirm that Hybrid NPs can penetrate the blood-brain barrier more efficiently and achieve enrichment at sites of cerebral ischemia-reperfusion injury.

[0079] Figure 12 These are neurological deficit scores in rats after treatment with different drugs.

[0080] The Longa score (Zea Longa neurological deficit score) is a classic quantitative standard for assessing the degree of neuromotor function deficit after MCAO in rats. A higher score indicates a more severe neurological deficit, while a lower score indicates better recovery of neuromotor function. This experiment included four groups: Sham group, MCAO model group, 7,8-DHF group, T7-liposomes group, Hybrid NPs group, and EVs-p53 siRNA group. The Longa score was used to assess the neurological function of rats in each group, clarifying the effects of different interventions on the recovery of neuromotor function after cerebral ischemia in rats.

[0081] The results are as follows Figure 12 As shown, the Sham group rats had a neurological function score of 0, with no neuromotor function deficits. Their limb movements were flexible and symmetrical, and turning and crawling were normal. Compared to the Sham group, the MCAO model group rats had a significantly higher Longa neurological function score, exhibiting obvious neuromotor function deficits. Some rats developed limb paralysis, indicating that the MCAO model was successfully established and that cerebral ischemia-induced damage led to severe neurological dysfunction. The scores of the 7,8-DHF group, T7-liposomes group, and EVs-p53 siRNA group were significantly lower than those of the MCAO group, with reduced limb flexion and circling deficits, indicating some degree of neurological function repair. The Hybrid NPs group rats had the lowest neurological function score among all groups, only 1-2 points, with limb movements essentially restored to normal, showing the best neuromotor function recovery.

[0082] Figure 13 The figures show the infarct volume of MCAO rats after different drug treatments; (A) is a TTC staining image, and (B) is a quantitative statistical graph of the infarct area of ​​brain tissue.

[0083] TTC can bind to dehydrogenases in living cells to form a red formazan product, while ischemic necrotic brain tissue, due to loss of enzyme activity, cannot show color and appears white. Therefore, TTC staining is a classic method for evaluating the area of ​​cerebral ischemia-infarction in rats. This experiment included the Sham group, MCAO model group, 7,8-DHF group, T7-liposomes group, Hybrid NPs group, and EVs-p53 siRNA group. Five days after MCAO model establishment, rat brain tissue was harvested, rapidly frozen, and stained with TTC to quantitatively analyze the degree of cerebral ischemia-infarction in each group.

[0084] The results are as follows Figure 13As shown, after TTC staining, the coronal sections of the brains of rats in the Sham group showed a uniform bright red color throughout the entire brain region, with no white ischemic infarct areas, indicating that the sham surgery did not cause ischemic damage to the brain tissue and the model background was normal. Compared with the Sham group, the MCAO model group showed a large area of ​​well-defined white ischemic infarcts in the right cerebral hemisphere of rats, while normal brain tissue remained red, indicating that the MCAO model was successfully established and that significant ischemic necrosis of brain tissue occurred. Compared with the MCAO model group, the white ischemic infarct areas in the brain sections of rats in the 7,8-DHF group, T7-liposomes group, and EVs-p53 siRNA group all showed varying degrees of reduction, while the area of ​​red normal brain tissue increased. The brain sections of the Hybrid NPs group showed only a small number of scattered white micro-infarct areas, with a significantly reduced ischemic infarct area (quantitative analysis showed approximately 10%), and the coverage of red normal brain tissue was close to that of the Sham group, indicating the best cerebral ischemia protection effect among all groups.

[0085] Figure 14 This is a graph showing the effects of different drug treatments on neurogenesis in MCAO rats.

[0086] This experiment detected neuronal apoptosis in the brain tissue of rats in the Sham, MCAO, 7,8-DHF, T7-liposomes, Hybrid NPs, and EVs-p53 siRNA groups by labeling neurons with TUJ1 and apoptotic cells with TUNEL. The results are as follows: Figure 14 As shown, extensive and continuous red TUJ1 fluorescence signals were observed in the brain tissue of rats in the Sham group. Neuronal morphology was intact and evenly distributed, with only a very small amount of scattered weak green fluorescence signals and no obvious neuronal apoptosis. In the MCAO group, the red TUJ1 fluorescence signal was significantly weakened and its continuity was disrupted in the brain tissue. Neuronal morphology was shrunken, and a large number of strong green TUNEL positive fluorescence signals were observed. Compared with the MCAO group, all treatment groups improved the fluorescence expression characteristics of brain tissue to varying degrees. The red TUJ1 fluorescence signal was somewhat restored in the brain tissue of the 7,8-DHF group, T7-liposomes group, and EVs-p53 siRNA group, while the green TUNEL positive fluorescence signal decreased. The Hybrid NPs group showed the most significant apoptosis inhibition effect, with significantly better intensity and continuity of the red TUJ1 fluorescence signal in the brain tissue than the other treatment groups, and the green TUNEL positive fluorescence signal was reduced to the lowest level among all treatment groups.

[0087] Figure 15 This is a graph showing the effects of different drugs on the blood-brain barrier integrity in MCAO rats.

[0088] The structural integrity of the blood-brain barrier depends on the stable maintenance of tight junctions between endothelial cells. ZO-1, as a core tight junction protein, is a key marker of blood-brain barrier impairment and increased permeability. This study included four groups: Sham, MCAO, 7,8-DHF, T7-liposomes, Hybrid NPs, and EVs-p53 siRNA. Brain tissue was harvested after establishing a rat MCAO model. Immunofluorescence staining was used to detect the expression distribution and fluorescence intensity of ZO-1 protein, evaluating the protective effect of different interventions on the integrity of the blood-brain barrier after ischemia. Results are as follows: Figure 15 As shown, ZO-1 in the brains of rats in the Sham group exhibited continuous and bright red fluorescence signals, indicating that the blood-brain barrier structure was intact and tight junction protein expression was normal in the sham-operated group. Compared with the Sham group, ZO-1 fluorescence in the MCAO group was significantly weakened and faded, and the continuity of the fluorescence signal was disrupted. The results indicate that the blood-brain barrier was severely damaged after MCAO modeling, with abnormal expression and distribution of the tight junction protein ZO-1 and a significant increase in barrier permeability. Compared with the MCAO model group, the ZO-1 fluorescence signal in the brains of rats in each treatment group recovered to varying degrees, with increased red fluorescence brightness and improved continuity, indicating a reduction in the degree of blood-brain barrier damage. The Hybrid NPs group showed the highest intensity and best continuity of red ZO-1 fluorescence in the brain, demonstrating the best protective effect on the blood-brain barrier.

[0089] Figure 16 These are images showing the effects of different drugs on the polarization staining of inflammatory cells in the brain tissue of MCAO rats.

[0090] This experiment used iNOS (green fluorescence, a pro-inflammatory phenotypic marker) and CD206 (red fluorescence, an anti-inflammatory phenotypic marker) for immunofluorescence staining, combined with laser confocal microscopy, to detect the polarization status of inflammatory cells in the brain tissue of rats in the Sham group, MCAO group, 7,8-DHF group, T7-liposomes group, Hybrid NPs group, and EVs-p53 siRNA group. The results are as follows: Figure 16 As shown, the Sham group rats were in a normal low-inflammatory state, with only a small amount of weak iNOS fluorescence signal and a relatively strong CD206 fluorescence signal. In the MCAO group, iNOS green fluorescence was significantly enhanced in brain tissue, while CD206 red fluorescence was significantly weakened, indicating that pro-inflammatory phenotype inflammatory cells were dominant. Compared with the MCAO group, all drug-treated groups could reverse the polarization trend of inflammatory cells in brain tissue to varying degrees, with iNOS green fluorescence signal downregulated and CD206 red fluorescence signal significantly increased compared to the model group. Pro-inflammatory phenotype cells decreased, while anti-inflammatory phenotype cells increased. The Hybrid NPs group exhibited the most significant polarization regulation effect, with iNOS green fluorescence signal in brain tissue decreasing to the lowest level and CD206 red fluorescence signal intensity significantly higher than the other drug-treated groups.

[0091] Figure 17 This is a graph showing the effects of different drugs on the activation of inflammatory cells in MCAO rats.

[0092] GFAP is a specific biomarker for astrocytes, and its activation is a core characteristic of reactive astrocyte proliferation. IBA1 is a marker molecule for microglia, and microglia activation is a key early event in neuroinflammation following cerebral ischemia. The activation levels of both can directly reflect the degree of neuroinflammation in brain tissue. This experiment included the Sham group, MCAO group, 7,8-DHF group, T7-liposomes group, Hybrid NPs group, and EVs-p53 siRNA group. After establishing the MCAO model, rat brain tissue was collected, and the expression and activation status of GFAP (green) and IBA1 (red) were detected by immunofluorescence staining to evaluate the regulatory effects of different interventions on neuroinflammation in the brain. The experimental results are as follows: Figure 17 As shown, compared with the Sham group, the MCAO group showed significantly enhanced GFAP and IBA1 fluorescence signals in the ischemic brain region of rats, with expanded red-green fluorescence coverage and significantly increased brightness, confirming the successful induction of severe neuroinflammatory responses in brain tissue by MCAO modeling. Compared with the MCAO model group, the GFAP and IBA1 fluorescence signals in brain tissue of each treatment group were weakened to varying degrees, glial cell activation was alleviated, and the level of neuroinflammatory inflammation decreased. Among them, the GFAP and IBA1 fluorescence signals in the brain of the Hybrid NPs group dropped to the lowest level among all groups, red-green fluorescence almost disappeared, and glial cell morphology returned to the resting state, similar to the Sham group. This group showed the most significant inhibitory effect on MCAO-induced neuroinflammatory reactions.

[0093] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing extracellular vesicle-liposome hybrid nanoparticles, characterized in that, Includes the following steps: (1) P53-siRNA was loaded into extracellular vesicles to obtain extracellular vesicles containing P53-siRNA, denoted as EVs-p53siRNA; P53-siRNA sequence: S: GGACAGCCAAGUCUGUUAUTT, as shown in SEQ ID NO: 1; AS: AUAAACAGACUUGGCUGUCCTT, as shown in SEQ ID NO: 2; (2) DSPE-TK-PEG2000-Mal was added to PBS buffer and dissolved by sonication; C-T7 peptide was added and reacted to obtain DSPE-TK-PEG2000-T7; (3) DOPC, DOTAP, Cholesterol, DSPE-PEG2000, DSPE-TK-PEG2000-T7 and 7,8-DHF were added to anhydrous ethanol, dissolved by sonication, and evaporated under reduced pressure to obtain a lipid film; the lipid film was hydrated, and the crude liposome suspension was treated with an ultrasonic water bath after hydration, and then extruded through a polycarbonate membrane to obtain 7,8-DHF-loaded liposomes, denoted as T7-liposomes; (4) Mix EVs-p53 siRNA with T7-liposomes and sonicate to freeze and thaw to obtain extracellular vesicle-liposome hybrid nanoparticles.

2. The preparation method according to claim 1, characterized in that, In step (1), extracellular vesicles are obtained by tangential flow ultrafiltration concentration, gradient centrifugation and ultracentrifugation extraction of P3-5 generation human umbilical cord mesenchymal stem cells; and / or, in step (1), P53-siRNA is loaded into EVs by electroporation.

3. The preparation method according to claim 1, characterized in that, In step (2), the molar ratio of DSPE-TK-PEG2000-Mal to C-T7 peptide is 1:1-1.1; and / or, in step (2), the reaction is carried out under nitrogen protection in the dark; and / or, in step (2), the reaction temperature is 20-30 ℃; and the reaction time is 20-25 h.

4. The preparation method according to claim 1, characterized in that, In step (3), the weight ratio of DOPC, DOTAP, Cholesterol, DSPE-PEG2000, DSPE-TK-PEG2000-T7 and 7,8-DHF is 26-28:8-10:4-6:1-3:2-4:4; and / or, in step (3), the reduced pressure evaporation conditions are set as follows: reduced pressure evaporation in a rotary evaporator at 25-35℃, with a rotation speed of 50-70 rpm.

5. The preparation method according to claim 1, characterized in that, In step (3), the hydration conditions are as follows: 7,8-DHF is dissolved in PBS to prepare a stock solution of 2-3 mg / mL, and the lipid membrane is hydrated with this solution at 650-680 rpm for 50-70 min at room temperature.

6. The preparation method according to claim 1, characterized in that, In step (3), the specific conditions for ultrasound are as follows: the ultrasound conditions are set to power of 65-70W, ultrasound water bath treatment for 8-12 min, ultrasound for 8-12 s, and stop for 8-12 s; and / or, in step (3), the polycarbonate membrane is a 300-500 nm and a 100-300 nm polycarbonate membrane.

7. The preparation method according to claim 1, characterized in that, In step (4), the volume ratio of EVs-p53 siRNA to T7-liposomes is 2-4:

1.

8. The preparation method according to claim 1, characterized in that, In step (4), the ultrasonic freeze-thaw treatment is as follows: the mixture of EVs-p53siRNA and T7-liposomes is rapidly immersed in liquid nitrogen for 20-40 s to freeze, and then immediately placed in a water bath at 36-38℃ for rapid thawing for 20-40 s. This is one complete freeze-thaw cycle. This cycle is repeated 2-4 times. Then the mixture is placed at room temperature to recover for 8-12 min, and ultrasonic treatment is performed for 4-6 min.

9. Extracellular vesicle-liposome hybrid nanoparticles prepared by the preparation method according to any one of claims 1 to 8.

10. The use of the extracellular vesicle-liposome hybrid nanoparticles as described in claim 9 in the preparation of drugs for treating cerebral ischemia-reperfusion injury.

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