Exosome liposome composite nanoparticle as well as preparation method and application thereof

Abstract

The invention discloses exosome and liposome composite nanoparticles and a preparation method and application thereof, and belongs to the technical field of biological medicine, the preparation method of the exosome and liposome composite nanoparticles comprises the following steps: performing membrane fusion on exosome derived from neural stem cells and liposome entrapped with Yulangsan polysaccharide to obtain the exosome and liposome composite nanoparticles. According to the composite nano-particles prepared by the method, Yulangsan polysaccharide and neural stem cell exosome are jointly applied, so that a remarkable synergistic treatment effect can be exerted on a multi-mechanism level, and the effect of treating cerebral arterial thrombosis can be remarkably improved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to exosome-liposome composite nanoparticles and a preparation method and application thereof. Background Art

[0002] Stroke, also known as cerebral infarction, is a common and complex cerebrovascular disease. Its pathogenesis involves multiple pathological processes, including oxidative stress, inflammatory response, and apoptosis. Stroke can be categorized by type as ischemic or hemorrhagic, with ischemic stroke accounting for over 85% of all strokes. To promptly restore blood flow to cerebral tissue and reduce infarct size, reperfusion therapies such as thrombolysis or mechanical thrombectomy are commonly used clinically. However, the process of blood flow restoration may induce secondary injury, namely ischemia-reperfusion injury, which manifests as further cellular damage and neurological dysfunction. The middle cerebral artery ischemia-reperfusion (MCAO / R) animal model is a promising approach to effectively simulate this secondary injury process.

[0003] Currently, treatments for stroke remain limited, particularly in the area of ​​neuroprotection. Therefore, exploring novel therapeutic strategies and drugs with neuroprotective effects based on the MCAO / R model has significant research value and application prospects for improving stroke prognosis. Summary of the Invention

[0004] In response to the problem that the above-mentioned prior art lacks therapeutic drugs for stroke, especially the lack of treatment methods for neuroprotection, the present invention provides an exosome-liposome composite nanoparticle (Exo-Lip), which has a neuroprotective effect and can significantly improve the effect of treating ischemic stroke.

[0005] On the one hand, the present invention provides a method for preparing exosome-liposome composite nanoparticles, comprising the following steps: fusing exosomes derived from neural stem cells with liposomes encapsulating Yulangsan polysaccharide to obtain the obtained exosomes.

[0006] After investigating many candidate active substances in the early stage, the inventors believe that neural stem cells have the potential for self-renewal and multidirectional differentiation, and can differentiate into neurons, astrocytes and oligodendrocytes. They are an important cell source for the repair and regeneration of the central nervous system. In addition, neural stem cells not only promote neural repair through direct differentiation, but also mediate intercellular communication by secreting exosomes, exerting a significant paracrine effect. Exosomes derived from neural stem cells (hereinafter referred to as Exo) are rich in a variety of bioactive substances, including neurotrophic factors, functional microRNAs and a variety of signal regulatory proteins. These functional molecules can show protective and repair effects on nerves in various pathological conditions such as neurodegenerative diseases, brain injury and spinal cord injury by inhibiting neuroinflammation, promoting neuronal axon regeneration, regulating neural plasticity and inhibiting cell apoptosis. In addition, exosomes, as a natural nano-delivery carrier, have cell-free properties, low immunogenicity, good biocompatibility and excellent cross-blood-brain barrier ability. They can be used as potential candidates to target brain injury areas, reduce nonspecific distribution and improve the accuracy of treatment.

[0007] On the other hand, based on the previous research of the inventors, considering that Yulangsan polysaccharide is a bioactive polysaccharide derived from natural plants, it has strong antioxidant, anti-inflammatory and immunomodulatory effects, and can help alleviate oxidative stress and inflammatory responses during stroke. However, previous studies have found that Yulangsan polysaccharide itself is difficult to effectively cross the blood-brain barrier, and its bioavailability in brain tissue is low, which significantly limits its neuroprotective potential. Liposomes, as a classic nano drug delivery system, have good biocompatibility, drug encapsulation capacity and surface functionalization modification potential. They can effectively prolong the circulation time of active ingredients in the body and promote the crossing of drugs across the blood-brain barrier to a certain extent.

[0008] Based on this research, the present invention utilizes a liposome-based encapsulation strategy to combine Yulangsan polysaccharide with neural stem cell exosomes to prepare composite nanoparticles, demonstrating significant synergistic therapeutic effects across multiple mechanisms. First, this combination effectively alleviates lipid metabolism disorders and lipid peroxidation following cerebral ischemia, reducing oxidative stress-related damage. Second, by promoting microglial polarization toward the M2 phenotype and inhibiting the proinflammatory response mediated by M1 microglia, it holistically modulates the local immune microenvironment and alleviates neuroinflammatory responses. Furthermore, the synergistic effect of exosomes and polysaccharides can directly protect neurons, inhibit apoptosis, and promote neural repair and functional reconstitution. It also helps restore cerebral blood perfusion in the damaged area and significantly reduces infarct volume. Thanks to the excellent blood-brain barrier-crossing ability of exosomes, the combined system enables targeted delivery and increases the effective concentration of drugs within brain tissue, further enhancing therapeutic efficacy and improving neurological prognosis after stroke.

[0009] In some embodiments, the membrane fusion is performed by reverse phase evaporation, extrusion, freeze-thaw, ultrasonic incubation, electrofusion or microfluidics.

[0010] In some embodiments, an ultrasonic incubation method is used to perform fusion according to the following steps: exosomes and liposomes are taken, placed in a PBS solution, and incubated after ultrasonication.

[0011] In some embodiments, the mass ratio of the exosomes to the liposomes is 3:1-1:3, for example, 3:1, 2:1, 1:1, 1:2, 1:3, preferably 1:1.

[0012] In some embodiments, the PBS solution has a concentration of 0.01-0.02 M and a pH of 7.2-7.6, preferably, a concentration of 0.01 M and a pH of 7.4.

[0013] In some embodiments, the concentration of the exosomes in the PBS solution is 0.01-0.2 μg / μL, preferably 0.2 μg / μL.

[0014] In some embodiments, the power of the ultrasound is 50-300W, preferably 80W.

[0015] In some embodiments, the ultrasonication time is 5-20 min, preferably 10 min.

[0016] In some embodiments, the ultrasound is performed in a cycle of 2-5 seconds on and 5-8 seconds off, preferably in a cycle of 3 seconds on and 7 seconds off.

[0017] In some embodiments, the incubation temperature is 4-42°C, preferably 37°C.

[0018] In some embodiments, the incubation time is 0.5-2 hours, preferably 1 hour.

[0019] In some embodiments, the neural stem cell-derived exosomes are prepared by the following method: performing cell culture on neural stem cells, collecting the culture supernatant, and obtaining exosomes by centrifugation.

[0020] In some of the methods for preparing exosomes, the neural stem cells are obtained by the following method: peripheral blood mononuclear cells are obtained, and Sendai virus containing transcription factors Oct4, Sox2, Klf4 and c-Myc is added for transfection, and then peripheral blood mononuclear cell expansion medium is added for culture, the cells are collected, hiNPCs medium is added, and the cells are inoculated on a plate covered with poly-D-lysine hydrobromide and laminin. After centrifugation, the cells are continued to be cultured until hiNPCs clones appear, thereby obtaining neural stem cells.

[0021] In some of the methods for preparing exosomes, the method for collecting the culture supernatant is: culturing neural stem cells in serum-free medium, discarding the old medium and replacing it with fresh medium when the cell density reaches 70% to 80%, placing the cells in a cell culture incubator and culturing for 48±12 hours, and then collecting the culture supernatant.

[0022] In some of the methods for preparing exosomes, the centrifugation conditions are: centrifugation at 300±100×g, 4±2°C for 20±5 min; taking the supernatant and centrifuging at 2000±100×g, 4±2°C for 20±5 min; taking the supernatant and filtering it through a 0.22μm filter membrane, and continuing to centrifuge at 10,000±100×g, 4±2°C for 1±0.1h; taking the supernatant and centrifuging it at 100,000±1000×g, 4±2°C for 90±5min, and discarding the supernatant to obtain the exosomes; preferably, centrifugation at 300×g, 4°C for 20 min; taking the supernatant and centrifuging it at 2000×g, 4°C for 20 min; taking the supernatant and filtering it through a 0.22μm filter membrane, and continuing to centrifuge at 10,000×g, 4°C for 1h; taking the supernatant and centrifuging it at 100,000g, 4°C for 90min, and discarding the supernatant to obtain the exosomes.

[0023] In some embodiments, the liposomes encapsulating Yulangsan polysaccharide are prepared by the following method: soybean lecithin, cholesterol and Tween 80 are dissolved in a first organic solvent, the organic solvent is removed by rotary evaporation to form a lipid film layer on the inner wall of the container, a second organic solvent and a PBS solution of the Yulangsan polysaccharide are added at the same time, ultrasonication is performed to form a uniform dispersion, the solvent is then evaporated by rotary evaporation to form a milky white or translucent dispersion, a PBS dispersion solution is added, rotary evaporation is continued until the solution becomes an aqueous suspension, the system is further dispersed by ultrasonication, and the macromolecules are removed by filtration to obtain the liposomes.

[0024] In some of the methods for preparing liposomes, the mass ratio of soybean lecithin, cholesterol and Tween 80 is 80-90:20-10:10-5, preferably 85:15:8.5.

[0025] In some of the methods for preparing liposomes, the first organic solvent is chloroform.

[0026] In some of the methods for preparing liposomes, the second organic solvent is diethyl ether.

[0027] In some of the methods for preparing liposomes, the volume ratio of the second organic solvent to the PBS solution of Yulangsan polysaccharide is 10-2:1, preferably 5:1.

[0028] In some of the methods for preparing liposomes, the ratio of the soybean lecithin to the second organic solvent is 5-15 mg:1 mL, preferably 10.2 mg:1 mL.

[0029] In some of the methods for preparing liposomes, the concentration of Yulangsan polysaccharide in the PBS solution of Yulangsan polysaccharide is 1-10 mg / mL, preferably 5 mg / mL.

[0030] In some of the methods for preparing liposomes, the time used for ultrasonication to form a uniform dispersion is 10-60 min, preferably 30 min.

[0031] In some of the methods for preparing liposomes, the time used for further dispersing the system by ultrasound is 5-30 minutes, preferably 10 minutes; and the process is carried out in a cycle of 3 seconds on and 7 seconds off.

[0032] In some of the methods for preparing liposomes, the pore size of the filter membrane used for filtering out macromolecules is 0.22-0.45 μm, preferably 0.22 μm.

[0033] In some of the methods for preparing liposomes, the obtained liposomes are stored at 4-37°C, preferably at 4°C.

[0034] In some embodiments, the Yulangsan polysaccharide is prepared by the following method: taking Yulangsan, adding ethanol-water solution to extract, adding water to the residue after the extract is concentrated, concentrating the water extract into an extract, adding ethanol, letting it stand and precipitate, and taking the precipitate to obtain Yulangsan polysaccharide.

[0035] In some of the methods for preparing Yulangsan polysaccharide, the volume percentage concentration of the ethanol aqueous solution is 50-80%, preferably 70%.

[0036] In some of the methods for preparing Yulangsan polysaccharide, the usage ratio of Yulangsan to ethanol aqueous solution is 1 kg:5-15 L, for example, 1 kg:10 L.

[0037] In some of the methods for preparing Yulangsan polysaccharide, the extraction with the ethanol aqueous solution is reflux extraction, and the extraction time is 1-5 hours, preferably 3 hours.

[0038] In some of the methods for preparing Yulangsan polysaccharide, the ratio of the amount of Yulangsan to the water added to the residue is 1 kg:5-15 L, for example, 1 kg:10 L.

[0039] In some of the methods for preparing Yulangsan polysaccharide, the residue is added to water for extraction by boiling, the extraction time is 1-5 hours, preferably 3 hours; the number of extractions is 2-4 times, preferably 3 times.

[0040] In some of the methods for preparing Yulangsan polysaccharide, the water extract is concentrated to a specific gravity of 1.0-1.2 at 50°C and then ethanol is added, preferably concentrated to a specific gravity of 1.1 at 50°C and then ethanol is added.

[0041] In some of the methods for preparing Yulangsan polysaccharide, the volume of ethanol added after the water extract is concentrated is 2-4 times, preferably 3 times, that of the extract.

[0042] In some of the methods for preparing Yulangsan polysaccharide, the precipitate is further dried under vacuum at 60-80° C. to obtain powdered Yulangsan polysaccharide.

[0043] On the other hand, the present invention also discloses exosome-liposome composite nanoparticles prepared by the above preparation method.

[0044] In some embodiments, the composite nanoparticles have a particle size of 100-200 nm, preferably 170-200 nm, for example, 190±5 nm.

[0045] In some embodiments, the exosomes are exosomes of neural stem cells obtained by induced differentiation of PBMCs.

[0046] On the other hand, the present invention also discloses the use of the above-mentioned exosome-liposome composite nanoparticles in the preparation of a method for preventing and / or treating ischemic stroke.

[0047] The composite nanoparticles exert their effects by protecting and repairing nerves, promoting recovery of cerebral blood flow, and / or reducing infarct volume.

[0048] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0049] The reagents and raw materials used in the present invention are commercially available.

[0050] The positive progress effect of the present invention is:

[0051] The composite nanoparticles obtained by the preparation method of the exosome-liposome composite nanoparticles of the present invention have excellent performance in the following aspects and have good clinical translation prospects:

[0052] 1. Good targeting and blood-brain barrier crossing ability: The Exo-Lip system constructed by the present invention combines the excellent natural targeting and good blood-brain barrier crossing ability of neural stem cell exosomes, which can achieve efficient delivery to brain damaged areas and significantly improve the enrichment efficiency of therapeutic drugs in brain tissue.

[0053] 2. Synergistically enhanced neuroprotection and repair effects: Neural stem cell exosomes and Yulangsan polysaccharide as composite nanoparticles work synergistically as a whole, which can effectively alleviate lipid metabolism disorders and lipid peroxidation, promote microglial M2 polarization, inhibit inflammatory response, protect neuronal cells, inhibit cell apoptosis, promote axon regeneration, and achieve comprehensive neuroprotection from multiple mechanisms.

[0054] 3. Promote cerebral blood flow recovery and reduce infarct volume: By improving the vascular microenvironment and protecting the function of the neurovascular unit, the composite nanoparticles of the present invention can effectively restore local blood perfusion of brain tissue, reduce secondary damage during ischemia-reperfusion, thereby significantly reducing the infarct area and promoting neurological function recovery.

[0055] 4. Improved system stability and bioactivity: Through the synergistic construction of Yulangsan polysaccharide and liposomes, the physical and chemical stability and in vivo circulation time of the composite nanoparticles are improved, and the overall bioactivity and therapeutic sustainability are enhanced.

[0056] 5. Good biocompatibility and safety: The composite nanoparticles of the present invention use ingredients of natural origin, have a mild preparation process, do not require exogenous carriers or harmful additives, have low immunogenicity, good biocompatibility and high safety, and are suitable for long-term intervention applications in neurological diseases such as stroke.

[0057] The present invention also provides a preparation method, biological function verification, and biosafety assessment method of the Exo-Lip, as well as its therapeutic application in preventing and / or treating ischemic stroke animal models.

[0058] The above-mentioned composite nanoparticle delivery system with stable structure, strong targeting and high biological activity is used for neuroprotective treatment of ischemic stroke, which has made important progress in translational medicine research of stroke and is expected to provide a new drug delivery platform and treatment ideas for the treatment of central nervous system diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 Transmission electron microscopy images;

[0060] Figure 2 The particle size and potential results of Exo, Lip, and Exo-Lip are shown;

[0061] Figure 3 The results of marker expression on the surface of cells, Exo, and Exo-Lip membranes are shown;

[0062] Figure 4 Mouse fluorescence imaging results measured by the mouse living imaging system;

[0063] Figure 5 Laser speckle contrast imaging of the brain of MCAO / R mice after treatment in different treatment groups;

[0064] Figure 6 This is a comparison of the brain tissues of mice in different treatment groups after TTC experiment;

[0065] Figure 7 Comparison of cerebral infarction area in mice of different treatment groups;

[0066] Figure 8 The data results after quantification of the water content in the brain of mice in different treatment groups;

[0067] Figure 9 Comparison of neurobehavioral scores of mice in different treatment groups;

[0068] Figure 10 This is a comparison of TUNEL staining results of brain tissues of mice in different treatment groups;

[0069] Figure 11 It is the gait footprint diagram;

[0070] Figure 12 The results of the open field test for mice;

[0071] Figure 13 This is a comparison of the staining results of brain tissues of mice in different treatment groups;

[0072] Figure 14 Comparison of immunofluorescence staining results of brain tissues of mice in different treatment groups;

[0073] Figure 15 Cytokine levels detected by ELISA;

[0074] Figure 16 is the level of lipid metabolism index;

[0075] Figure 17 This is a comparison of the Oil Red staining results of brain tissues of mice in different treatment groups;

[0076] Figure 18 Comparison results of Oil Red staining of brain tissues of mice in different treatment groups;

[0077] Figure 19 is the expression level of lipid metabolism-related genes in the brain tissues of mice in different treatment groups;

[0078] Figure 20Comparison of HE staining results of mouse tissues in different treatment groups;

[0079] Figure 21 are the blood routine indicators of mice in different treatment groups;

[0080] Figure 22 Heat map of RNA sequencing results for different groups of mice;

[0081] Figure 23 Volcano plot of RNA sequencing results for different groups of mice;

[0082] Figure 24 This is the result of KEGG pathway enrichment analysis;

[0083] Figure 25 Gene set enrichment analysis results of important pathways. DETAILED DESCRIPTION

[0084] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0085] Unless otherwise specified, the PBS used in the following examples is 0.01 M phosphate buffered saline, pH 7.4.

[0086] Example 1: Preparation of Exosome-liposome composite nanoparticles (Exo-Lip)

[0087] An exosome-liposome composite nanoparticle (Exo-Lip) is prepared by the following method:

[0088] 1. Induced neural stem cells

[0089] Take 20 mL of human peripheral blood, dilute with an equal volume of PBS, and divide into four aliquots. Carefully add the blood sample at a 45° angle to the upper layer of an equal volume of mononuclear cell separation buffer. Centrifuge at 750 × g for 30 minutes at 25°C. Discard the supernatant, and transfer the white cloudy layer to two new 15 mL centrifuge tubes. Add 10 mL of PBS to each tube, resuspend the cells, and centrifuge at 300 × g for 10 minutes at 4°C. Discard the supernatant, add 10 mL of PBS to each tube, resuspend the cells, and centrifuge at 300 × g for 10 minutes at 4°C. Discard the supernatant and collect the PBMCs.

[0090] PBMCs were collected at a rate of 0.5-1×10 6Cells were seeded at a density of 100 cells / mL in a 6-well plate and cultured in PBMCs expansion medium, with the medium changed every two days. Culture conditions were 5% CO2, 37°C, and relative humidity maintained. On the third day of culture, cells and medium were transferred to a new 15 mL centrifuge tube and centrifuged at 200 g for 3 minutes to collect PBMCs. After counting with trypan blue, the cells were counted at 0.5-1 × 10 6 The cells were seeded at a density of 0.5-1×10 cells / mL in a 6-well plate and the medium was changed every 2 days. 6 The PBMCs were inoculated at a density of 100 cells / mL and cultured for 14 days.

[0091] On the 14th day of cell expansion, 6 × 10 6 cells, add 6 mL of PBMCs expansion medium, which contains 8 × 10 6 1 mL of Sendai virus was inoculated into 6 wells of a 6-well plate. 12 hours after transfection, 1 mL of PBMC expansion medium was added to each well. On the 3rd day after transfection, cells were harvested and hiNPC culture medium was added. The cells were cultured at a density of 1×10 5 Cells were seeded at a density of 100 cells / mL in a 12-well plate coated with poly-D-lysine hydrobromide and laminin, centrifuged at 200 × g for 30 minutes at 25°C, and then cultured in a 37°C, 5% CO2 incubator. The medium was replaced with hiNPC culture medium every other day. Approximately 11 days after transfection, when hiNPC foci appeared, neural stem cells were obtained.

[0092] 2. Preparation of neural stem cell exosomes (Exo)

[0093] The neural stem cells obtained above were cultured in serum-free medium. When the cell density reached 70% to 80%, the old medium was discarded and replaced with fresh medium. The cells were cultured in a cell culture incubator for 48 hours. After 48 hours of culture, the supernatant was collected. The collected culture supernatant was placed in a 50 mL centrifuge tube and centrifuged at 300 × g and 4 ° C for 20 minutes to remove cells. The supernatant was taken from a 50 mL centrifuge tube and centrifuged at 2000 × g and 4 ° C for 20 minutes to remove debris. The supernatant was filtered through a 0.22 μm filter membrane into an ultracentrifuge tube and centrifuged at 10,000 × g and 4 ° C for 1 hour. The supernatant was taken from an ultracentrifuge tube and centrifuged at 100,000 × g and 4 ° C for 90 minutes. The supernatant was discarded and the precipitate was resuspended in 100 μL of pre-chilled PBS (0.01 M, pH = 7.4) to obtain neural stem cell exosomes (Exo). It was stored in a -80 ° C refrigerator for future use.

[0094] 2. Preparation of liposomes encapsulating Yulangsan polysaccharide (Lip) by thin film hydration method

[0095] 2.1 Preparation of Yulangsan Polysaccharide (YLSP):

[0096] Take 10 kg of dried roots of Yulangsan, cut into small pieces, add 100 L of 70% ethanol aqueous solution and reflux extract once for 3 hours, recover ethanol, add 100 L of distilled water to the concentrated residue and boil it 3 times for 3 hours each time, combine the water extracts, filter, and vacuum concentrate the filtrate to a specific gravity of 1.1 (50°C), add 3 times the amount of 95% ethanol and precipitate for 24 hours, filter, take the precipitate, and dry it in a vacuum oven at 70°C to obtain powdered Yulangsan polysaccharide for later use.

[0097] 2.2 Preparation of Yulangsan polysaccharide liposomes (Lip)

[0098] Take soybean lecithin, cholesterol and Tween 80, the amounts of which are 102 mg, 18 mg and 10.2 mg (85:15:8.5) respectively, and dissolve them ultrasonically in 7 mL of chloroform. After complete dissolution, the organic solvent is removed by rotary evaporation and a lipid film is formed on the wall of the flask.

[0099] The lipid film was then redissolved in 10 mL of ether and 2 mL of PBS solution containing the drug to be encapsulated, Yulangsan polysaccharide (YLSP, 5 mg / mL), was added at the same time. The two systems were ultrasonicated for 30 min until they were mixed into a uniform dispersion. At this time, a stable W / O emulsion was obtained. The emulsion was placed in a rotary evaporator and distilled under reduced pressure at 35 ° C. With the loss of a large amount of organic solvent and water, a viscous gel state gradually appeared. When a milky white or translucent dispersion was formed, 6 mL of PBS buffer was added as a dispersion liquid. The solution was continued to be distilled under reduced pressure at 35 ° C in a rotary evaporator. Over time, it gradually became an aqueous suspension. The suspension was then placed on ice and ultrasonicated for 10 min at a power of 200 W, according to a cycle of starting 3 s and stopping 7 s, to make the system more uniformly dispersed. After removing excessively large molecules with a 0.22 μm pore size filter membrane, the solution was stored at low temperature (e.g., 4 ° C) to obtain Yulangsan polysaccharide liposomes (Lip) for later use.

[0100] 2.3 Preparation of exosome-liposome composite nanoparticles (Exo-Lip) by ultrasonic incubation

[0101] Take 1 portion of Exo (100 μg protein) and 100 μg of Lip, mix them evenly, and then dilute to 500 μL with PBS. After ultrasonication at 80 W power for 10 minutes (operated at intervals of 3 seconds on and 7 seconds off), incubate at 37°C for 1 hour, use an ultrafiltration centrifuge tube with a molecular weight cutoff of 100 kD, centrifuge at 10,000g for 10 minutes, and wash with PBS 2-3 times to remove free Exo, Lip and small molecule impurities, retain the Exo-Lip complex, and obtain exosome liposome composite nanoparticles (Exo-Lip).

[0102] 3. Test and identification

[0103] Transmission electron microscopy, ZeTa particle size potential analyzer and Western blot were used to measure the exosomes derived from the neural stem cells isolated above.

[0104] The transmission electron microscopy determination method is as follows: the obtained exosome-liposome composite nanoparticles are diluted and dropped onto a carbon film-coated copper mesh, incubated for about 1 minute, and the residual liquid is absorbed with filter paper. After 2% (w / v) phospho-triglycine negative staining for 1 minute, the residual liquid is absorbed with filter paper, and after drying, the morphology of the drug delivery system is observed using a transmission electron microscope (JEM1200EX, Japan).

[0105] ZeTa particle size potential analysis method is to use Malvern particle size analyzer (Zetasizer Nano ZS, England) to measure the particle size and potential of nano-exosome-liposome composite nanoparticles.

[0106] Western blot analysis involves protein extraction from the exosome-liposome composite nanoparticle sample and quantification using the BCA assay. Equal amounts of protein (e.g., 20–30 μg) are then separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane. The membrane is blocked with 5% skim milk powder or BSA for 1 hour at room temperature. Primary antibodies (CD9, CD63, TSG101, and Calnexin, diluted 1:1,000) are then added and incubated overnight at 4°C. After washing, the membrane is incubated with the corresponding HRP-conjugated secondary antibody (e.g., 1:5,000) at room temperature for 1 hour. After washing with TBST, the membrane is developed using ECL luminescence solution, and protein bands are detected using a gel imaging system.

[0107] The results are as follows Figure 1-3 As shown, Figure 1 The following are transmission electron micrographs (TEM images), where A, B, and C represent the morphological images of Exo, Lip, and Exo-Lip, respectively. The results show that Exo exhibits a round or typical saucer-shaped shape, Lip appears as spherical vesicles with a uniform membrane structure, and Exo-Lip appears as spherical nanoparticles with an enlarged size and a slightly roughened surface, indicating successful fusion of the two.

[0108] Figure 2Figures 1 and 2 show the particle size and potential results for Exo, Lip, and Exo-Lip, with A showing the particle size and B showing the potential. The particle size results show that the average hydrated particle sizes of Exo, Lip, and Exo-Lip are 138.43±23.76nm, 144.50±0.70nm, and 189.93±3.45nm, respectively. Zeta potential analysis indicates that all three nanoparticles are negatively charged, with the surface potential of Exo-Lip lying between that of Exo and Lip, reflecting good hybridization efficiency and colloidal stability.

[0109] Figure 3 Figure 2 shows the expression of CD9, CD63, TSG101, and Calnexin on the membrane surfaces of cells, Exo, and Exo-Lip. This Western blot analysis further validates the quality of exosome isolation and the preservation of their structure in Exo-Lip. Both Exo and Exo-Lip express the classic exosomal markers CD9, CD63, and TSG101, while the endoplasmic reticulum protein Calnexin is not detected, indicating the absence of organelle contamination and the preservation of exosome structure.

[0110] The above experiments show that this example successfully isolated and obtained exosomes from neural stem cells, encapsulated Yulangsan polysaccharide in liposomes, and successfully assembled the two together.

[0111] Effect Experiment Example 1:

[0112] 1. Establishment of an Animal Model of Cerebral Ischemia-Reperfusion Injury

[0113] In this example, MCAO / R (Middle Cerebral Artery Occlusion / Reperfusion) animals were used to establish an animal model of cerebral ischemia-reperfusion injury.

[0114] This study used SPF-grade healthy male C57 mice (weighing 20-25 g, aged 7-8 weeks). Before the experiment, all mice were adapted to a 12-h light-dark alternating environment for 7 days and had free access to food and water. The mice were randomly divided into 5 groups: sham group (Sham), MCAO / R+PBS group (PBS), MCAO / R+Exo group (Exo), MCAO / R+Lip group (Lip), and MCAO / R+Exo-Lip group (Exo-Lip), with 20 mice in each group. Except for the sham group, the mice in other groups were subjected to MCAO / R animal model construction, while the sham group only underwent preoperative anesthesia and vascular isolation without ligation or introduction of sutures. Three days before the MCAO / R animal model was constructed, within 1 hour after the operation, each group was injected with drugs through the tail vein, that is, no drugs were injected in the sham operation. Each mouse in the MCAO / R+PBS group was injected with 100 μL PBS, once a day for 3 consecutive days; the treatment method of the MCAO / R+Exo group was to prepare the Exo prepared in Example 1 with PBS into 1 mg / mL neural stem cell-derived exosomes, and then inject 100 μL into each mouse, once a day, for 3 consecutive days; the treatment method of the MCAO / R+Lip group was to prepare the Lip prepared in Example 1 into 1 mg / mL liposomes encapsulating Yulangsan polysaccharide with PBS, and then inject 100 μL into each mouse, once a day, for 3 consecutive days; the treatment method of the MCAO / R+Exo-Lip group was to prepare the Exo-Lip nanoparticles prepared in Example 1 with a mixed concentration of 1 mg / mL, and then inject 100 μL into each mouse, once a day, for 3 consecutive days.

[0115] On the third day, one hour after drug administration, the MCAO / R model was established. Anesthesia was performed using an intraperitoneal injection of 1% sodium pentobarbital (5 mL / kg). After full anesthesia, the mice were placed in the supine position, their heads and limbs immobilized, the neck hair shaved, and the neck disinfected with iodine. A midline cervical incision was made with ophthalmic scissors, and the skin and soft tissue were pulled apart with a small retractor to expose the vessels. The left common carotid artery (CCA) and external carotid artery (ECA) were isolated. A slipknot was tied at the CCA to avoid irritation of the vagus nerve and trachea, which accompany the common carotid artery. The distal end of the ECA was tied with a knot and ligated, the proximal end of the ECA near the bifurcation, and a slipknot was tied midway through the ECA with suture. A slipknot was tied around the ECA proximal to the CCA bifurcation. The distal end of the ECA was tied with a knot near the head and secured with tape to provide gentle traction. A slipknot was tied proximal to the internal carotid artery (ICA). A suture bolt of appropriate weight for the mouse is used. Using ophthalmic scissors, a small incision is made at a 45-degree angle toward the ECA bifurcation between the first and second wires at the distal end of the ECA. Do not cut the incision. Insert the suture bolt from the opening toward the ECA bifurcation and secure it with a second wire to prevent slippage. Loosen the proximal wires at the ECA, sever the distal wires, and slowly insert the suture bolt toward the ICA. Stop insertion immediately if resistance is encountered, and suture the wound. Remove the suture bolt 2 hours after embolization. Maintain the mouse's body temperature at approximately 37°C using a thermostatic pad during surgery. Postoperatively, place the mouse on a warming blanket until awake.

[0116] Thus, the animal model of middle cerebral artery ischemia-reperfusion (MCAO / R) was obtained.

[0117] 2. Testing the brain targeting ability of Exo-Lip in mice

[0118] The distribution of Exo, Lip, and Exo-Lip in C57 mice was measured using a mouse in vivo imaging system.

[0119] Ordinary C57 mice were injected through the tail vein with 100 μL of DiR-labeled Exo, Lip, and Exo-Lip at a concentration of 1 mg / mL. At 3, 6, 12, and 24 hours after administration, an imaging system was used to collect the distribution of the drug in vivo and imaging of major organs. During the treatment process, the in vivo distribution of nanoparticles was first monitored in real time by DiR fluorescent labeling. The specific method is: at 3, 6, 12, and 24 hours after administration, fluorescence imaging was performed using a small animal in vivo imaging system to observe the dynamic distribution of nanoparticles in vivo. At the endpoint time (24 hours), the mice were killed, and the main organs (heart, liver, spleen, lung, kidney, and brain) were taken. After rinsing the surface blood with PBS under dark conditions, in vitro tissue imaging was performed to further evaluate the biodistribution of nanocarriers in various tissues.

[0120] The results are as follows Figure 4Figure 2 shows the fluorescence images of DiR-labeled Exo, Lip, and Exo-Lip in the isolated brain at 3, 6, 12, and 24 hours after injection, and Figure 2 shows the biodistribution of DiR-labeled Exo, Lip, and Exo-Lip in MCAO / R mice over time. In vivo fluorescence imaging revealed that Exo-Lip rapidly distributed throughout multiple tissues after injection, forming a significant fluorescence signal in the brain. Further in vitro brain tissue imaging and fluorescence quantitative analysis revealed that Exo-Lip accumulation in the brain peaked 12 hours after injection, significantly exceeding that of the Exo or Lip treatment groups alone.

[0121] This result demonstrates that Exo-Lip possesses excellent brain-targeted delivery and tissue penetration in vivo. This enhanced accumulation is likely due to the blood-brain barrier penetration mediated by peptides and transmembrane proteins on the exosome membrane surface, as well as the stable encapsulation and protection of the liposome structure for exosomes, achieving synergistic optimization of the structural integrity and delivery efficiency of the drug delivery system.

[0122] 3. Testing the effects of Exo-Lip on cerebral blood flow recovery, cerebral infarction area, brain edema, and neurobehavioral scores in mice

[0123] 1. Effect of Exo-Lip on cerebral blood flow recovery in mice

[0124] 24 hours after the MCAO / R animal model surgery, the cerebral blood perfusion of the mice in the Sham group, PBS group, Exo group, Lip group and Exo-Lip group was monitored in real time using a laser speckle blood flow imaging system.

[0125] The results are as follows Figure 5 As shown, Figure 5 These are laser speckle contrast imaging images of the brains of MCAO / R mice after treatment with different treatment groups. The results show that there were significant areas of reduced cerebral perfusion in the MCAO / R model group, while treatment with Exo or Lip alone only improved cerebral blood flow to a certain extent. The Exo-Lip treatment group was able to significantly restore cerebral blood flow in the ischemic area, demonstrating stronger cerebral blood flow reconstruction ability and neuroprotective potential.

[0126] 2. Effect of Exo-Lip on cerebral infarction area in mice

[0127] Twenty-four hours after surgery to establish the MCAO / R model, brain tissue was dissected from mice in the sham, PBS, Exo, Lip, and Exo-Lip groups. The brain tissues were sectioned, stained with TTC, and photographed. TTC (2,3,5-triphenyltetrazolium chloride) is a lipid-soluble, photosensitive compound that reacts with dehydrogenases in normal tissue, producing a red color. However, dehydrogenase activity in ischemic tissue is reduced, preventing the reaction and resulting in a pale color. Therefore, TTC staining is used to visualize infarct lesions and infarct volume. Non-infarcted areas stain red, while infarcted areas appear white. Brain tissue from each treatment group was collected and frozen at -20°C for 10 minutes. Sections were then cut coronally into 1 mm thick sections and stained with 2% TTC in PBS at 37°C for 15 minutes. Following staining, the sections were fixed with 4% paraformaldehyde at 4°C for 24 hours. Areas without red staining represent infarcted areas. Infarct volume was calculated for each group using ImageJ software.

[0128] The results are as follows Figure 6-7 As shown, Figure 6 This is a comparison of the brain tissues of mice in different treatment groups after TTC experiment. Figure 7 The results are the comparison of cerebral infarction area in mice treated with different methods. The infarction areas of the sham group, PBS group, Exo group, Lip group and Exo-Lip group were 22.64±5.60%, 12.07±3.46%, 7.43±1.52% and 5.13±1.43% in the PBS group, Exo group, Lip group and Exo-Lip group, respectively. Figure 6-7 The results clearly showed that no infarction occurred in the sham operation group, and no white infarct area was observed in the brain tissue, while obvious white infarct area appeared in the PBS group. This indicated that the MCAO / R mouse model was successfully constructed. Compared with the model group, the injection of Exo and Lip respectively could effectively reduce the infarct area, and Exo-Lip reduced it more, indicating a synergistic therapeutic effect.

[0129] 3. Effect of Exo-Lip on brain edema in mice

[0130] 24 hours after surgery, the brain tissue of mice in the sham, PBS, Exo, Lip, and Exo-Lip groups was dissected and isolated. The brain tissue was quickly removed, the surface liquid was aspirated, and the wet weight was measured. The brain tissue was then dried in a 100-110°C oven for 24 hours, and the dry weight was then measured. Brain water content was calculated as follows: water content (%) = (wet weight - dry weight) / wet weight × 100%.

[0131] The results are as follows Figure 8 As shown, Figure 8The data results of quantifying the water content in the brains of mice in different treatment groups showed that the brain tissue edema of mice in the PBS group was obvious compared with the Sham group, while the degree of edema in the Exo-Lip group was significantly reduced, indicating that the injection of Exo-Lip significantly reduced the brain tissue edema of mice.

[0132] 4. Effects of Exo-Lip on Neurobehavioral Scores of Mice

[0133] To assess neurological injury, PBS, Exo, Lip, or Exo-Lip were intravenously injected at a dose of 100 μg (1 mg / mL) per mouse. Twenty-four hours after administration and model establishment, mice were evaluated according to the Zea-Longa scoring system, and neurobehavioral and deficit scores were recorded for each group of MCAO / R mice. The Zea-Longa scoring system is 0: no neurological signs; 1: inability to fully extend the contralateral forelimb; 2: turning in circles toward the contralateral side while walking; 3: falling toward the contralateral side while walking; 4: inability to walk spontaneously and loss of consciousness; and 5: death.

[0134] The results are as follows Figure 9 As shown, Figure 9 The results show that the neurobehavioral scores of mice in different treatment groups were compared. The results showed that compared with the Sham group, the neurological function scores of mice in the PBS group were significantly increased; compared with the PBS group, the neurological scores of the Exo-Lip treatment group showed a significant downward trend, indicating that the neurobehavioral ability of mice was significantly improved after the injection of Exo-Lip.

[0135] 4. Testing the effect of Exo-Lip on apoptosis of mouse brain tissue cells

[0136] The level of apoptosis in brain tissue was assessed by TUNEL staining, specifically: the mice were decapitated after treatment, the brains were quickly removed, and fixed in 4% paraformaldehyde fixative at 4°C for 24 hours. Subsequently, gradient dehydration, paraffin embedding, and slices were made into 5μm thick serial tissue sections. After routine dewaxing and hydration, the sections were treated with a TUNEL staining kit according to the instructions. The brief steps include: the sections were treated with proteinase K (20μg / mL, room temperature for 20 minutes) to permeabilize the cell membrane, washed with PBS, and TUNEL reaction solution was added dropwise, and incubated in a 37°C incubator for 60 minutes in the dark. After staining, the cell nuclei were counterstained with DAPI, and the green fluorescence (TUNEL-positive cells) and blue fluorescence (DAPI-stained nuclei) signals were observed using a fluorescence microscope.

[0137] The results are as follows Figure 10 As shown, Figure 10The following figure compares TUNEL staining results for mouse brain tissue from different treatment groups. The results show that the number of TUNEL-positive cells in brain tissue was significantly reduced after Exo-Lip treatment. Compared with the PBS group and other control groups, Exo-Lip has a more pronounced effect in reducing cell apoptosis, indicating its potential application in inhibiting brain tissue apoptosis.

[0138] 5. Testing the improvement of mouse behavior by Exo-Lip

[0139] 24 hours after MCAO / R administration, mice from each group were placed in the same position in an open field. The distance traveled, as well as the distance and time spent in the edge and center zones, were recorded over a 10-minute period. Specifically, the left paw of the mice was smeared with black ink, and the right paw was smeared with red ink. The mice were then allowed to start from the same starting point on an A4 sheet of white paper and allowed to walk freely. The gait behavior of the mice was observed and recorded. The open field test was conducted in an open field box 50 cm long, 50 cm wide, and 50 cm high, with a floor divided into a center zone and an edge zone. Initially, the treated MCAO mice were placed in the same position on the edge zone of the open field box floor, and their activity was observed over a 10-minute period. The software recorded the total distance traveled, the distance traveled in the center zone, the percentage of distance traveled in the center zone, and the percentage of time spent in the center zone. After each mouse was tested, the inside of the box was wiped with 75% alcohol to eliminate any residual body odor. The box was allowed to dry before the next mouse was tested.

[0140] The results are shown in the following table and Figure 11-12 As shown, Figure 11 is the gait footprint diagram, Figure 12 The figures are the results of the open field test for mice, where A is a representative motion trajectory diagram of mice in different treatment groups in the open field test; B is a comparison of the time that mice in different treatment groups stayed in the central area in the open field test; C is a comparison of the total distance moved by mice in different treatment groups in the open field test; D is a comparison of the distance moved in the central area by mice in different treatment groups in the open field test; and E is a comparison of the percentage of distance moved in the central area by mice in different treatment groups in the open field test.

[0141] Table 1. Results of open field test in each group of mice

[0142]

[0143] The results showed that mice in the PBS group exhibited decreased activity in the open field, decreased exploration of the central zone, and increased wandering in the peripheral zone, reflecting significant motor impairment and anxiety-like behaviors. In contrast, the Exo and Lip treatment groups showed only slight improvements, while the Exo-Lip treatment group showed a significant increase in the total movement path, with significantly longer entries into the central zone and longer stays, suggesting a significant advantage in promoting motor function recovery and alleviating anxiety-like behaviors.

[0144] VI. Testing the Effect of Exo-Lip on Neurons after Cerebral Infarction in Mice

[0145] To further verify the effects of different groups on the pathological levels of damaged brain tissue, brain tissue from the MCAO / R animal model 24 hours after surgery was obtained and stained with hematoxylin and eosin (HE) and Nissl stains. Images of the injury margins were captured and magnified for display. The specific hematoxylin and eosin (H&E) staining method was as follows: After the animal experiment, the mice were anesthetized, decapitated, and the brains removed. After washing with saline, the brain tissue was fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned. After dewaxing the paraffin sections, they were stained with hematoxylin and eosin, dehydrated, and sealed with neutral resin. Under a microscope, cell nuclei appear blue and cytoplasm red. Nissl staining was performed as follows: After dewaxing the paraffin sections, they were stained with 0.5% toluidine blue, dehydrated, and sealed with neutral resin, and then observed under a microscope. Nissl bodies within neurons appear as dark blue granules, while cell nuclei appear light blue against a light blue background.

[0146] The results are as follows Figure 13 As shown, Figure 13 The red and blue pictures in the middle are comparisons of HE staining and Nissl staining results of brain tissues of mice in different treatment groups, respectively.

[0147] H&E staining results showed that in the PBS-treated group, MCAO / R induced significant damage to the cortical and hippocampal structures, manifested as typical ischemic injury characteristics such as neuronal morphological atrophy, disordered arrangement, and unclear cell outlines. In contrast, the structural integrity of the brain tissue in the Exo-Lip-treated group was significantly better than that in the PBS group, with more orderly neuronal arrangement and a significantly reduced number of atrophic and necrotic cells, suggesting that it has a good protective effect on neural structure. At the same time, the functional status of neurons was observed by Nissl staining. In the PBS group, the number of Nissl bodies in the hippocampus was significantly reduced, indicating extensive neuronal damage and metabolic disorders. After Exo-Lip treatment, the number of Nissl bodies in the hippocampus of mice was significantly retained, the neuronal morphology was plump, and the cell body staining was clear, indicating that Exo-Lip can effectively inhibit ischemia-induced neuronal degeneration and maintain the normal structure and function of neurons.

[0148] VII. Exo-Lip regulates M2 microglial polarization and inflammatory response in MCAO / R mice

[0149] Immunohistochemical analysis was performed to examine the expression of M1 (CD16 / 32) and M2 (CD206) microglia in the brain tissue of MCAO / R model mice. Furthermore, the levels of these cytokines were measured in brain tissue homogenates to reflect the regulatory effect of Exo-Lip on the local brain tissue immune microenvironment. Immunohistochemical analysis was performed to examine the expression of CD16 / 32, a marker for M1 microglia, and CD206, a marker for M2 microglia, in the brain tissue of MCAO / R model mice. Postoperative brain tissue was obtained from mice, fixed with 4% paraformaldehyde, embedded, and sectioned. The tissue was stained with the corresponding primary antibodies (e.g., CD16 / 32 and CD206, 1:200) and the distribution and expression levels of positive cells were analyzed using DAB staining and light microscopy.

[0150] The levels of proinflammatory cytokines TNF-α and IL-6 and anti-inflammatory cytokines TGF-β and IL-10 in the brain tissue of MCAO / R model mice were then detected by ELISA. The specific method is: the corresponding brain tissue was prepared into a homogenate, and the levels of proinflammatory factors TNF-α, IL-6 and anti-inflammatory factors TGF-β, IL-10 were quantitatively detected by ELISA to further reflect the regulatory effect of Exo-Lip on the expression of local inflammatory factors. The experiment used a commercial ELISA kit (Fankewei) and was operated according to the instructions. The absorbance value was read using a microplate reader at a wavelength of 450nm, and the concentration of each factor was calculated using a standard curve.

[0151] The results are as follows Figure 14-15 As shown, Figure 14 The following is a comparison of the immunofluorescence staining results of brain tissues of mice in different treatment groups (DAPI, blue, cell nuclei; CD16 / 32, green, M1 type; CD206, red, M2 type). The results showed that the expression of CD16 / 32 was significantly enhanced in the PBS group, while the expression of CD206 was significantly restricted, suggesting that MCAO / R injury induced a pro-inflammatory response dominated by the M1 type. In contrast, the Exo and Lip treatment groups weakened the expression of CD16 / 32 and enhanced the level of CD206 to a certain extent, indicating that they have a partial regulatory effect on the polarization state of microglia. In particular, the expression of CD206 in the Exo-Lip group was greatly increased, while the expression of CD16 / 32 was significantly downregulated, indicating that Exo-Lip has a stronger ability to promote the transformation of microglia to the anti-inflammatory and reparative M2 phenotype.

[0152] Figure 15The cytokine levels were detected by ELISA, where AD represents the levels of pro-inflammatory cytokines TNF-α and IL-6 and anti-inflammatory cytokines TGF-β and IL-10, respectively. The results showed that the levels of TNF-α and IL-6 in the PBS group were significantly increased, while the levels of TGF-β and IL-10 were significantly decreased, reflecting the systemic pro-inflammatory environment caused by MCAO / R injury. Exo and Lip treatment inhibited the excessive secretion of TNF-α and IL-6 to a certain extent, and slightly increased the levels of anti-inflammatory factors. The Exo-Lip treatment group had the most significant effect in reducing TNF-α and IL-6 and increasing TGF-β and IL-10, indicating that it has superior anti-inflammatory and immunomodulatory abilities.

[0153] The results showed that Exo-Lip not only significantly promoted the transformation of microglia from M1 to M2 phenotype in vivo, but also systematically and locally modulated the expression profile of inflammatory factors, thereby creating an anti-inflammatory microenvironment conducive to neuroprotection and tissue repair. These results further demonstrate the potential application value of Exo-Lip in immune intervention after cerebral ischemia-reperfusion injury.

[0154] 8. Testing Exo-Lip to Alleviate MCAO / R-Induced Lipid Metabolism Disorders and Lipid Peroxidation

[0155] 1. Lipid metabolism

[0156] Lipid metabolism disorders and lipid peroxidation play a key role in ischemia-reperfusion injury. To further evaluate the intervention effect of Exo-Lip on lipid homeostasis disruption caused by MCAO / R, a number of lipid metabolism-related indicators were detected in the MCAO / R model. The specific method is as follows: 24 hours after reperfusion, mice were killed, the ischemic hemisphere brain tissue was obtained, washed with PBS and homogenized at low temperature, and lipid metabolism indicators were measured using corresponding commercial kits, including triglyceride (TG), free fatty acid (FFA), malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) activity as evaluation indicators of lipid peroxidation damage and antioxidant defense capacity. The operation was carried out according to the kit instructions, and the absorbance value was read at a specific wavelength using a microplate reader.

[0157] The results are as follows Figure 16 As shown, Figure 16are lipid metabolism indicators, among which AD are the levels of triglyceride (TG), free fatty acid (FFA), malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), respectively. The results showed that the levels of triglyceride (TG) and free fatty acid (FFA) in the brain tissue of mice in the PBS group were significantly increased, indicating obvious lipid metabolism disorder. Exo and Lip treatment can partially reduce the levels of TG and FFA, and the effect of Exo-Lip treatment is the most significant. At the same time, the lipid peroxidation products malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) were significantly increased in the PBS group, while significantly decreased in the Exo-Lip group, indicating that Exo-Lip significantly alleviates lipid peroxidation damage.

[0158] 2. Histological Analysis

[0159] Histological analysis was further performed using Oil Red O staining. The brains (ischemic hemisphere) were quickly removed after mouse sacrifice, rinsed with PBS, and fixed in 4% paraformaldehyde for 4 h. The tissues were then dehydrated in 30% sucrose solution at 4°C until the tissue sank. After dehydration, the tissues were embedded in optical coherence tomography (OCT), and 10-μm-thick frozen sections were prepared using a cryostat and mounted on pretreated slides. The sections were air-dried at room temperature for 10 min and then stained with 0.5% Oil Red O stain (prepared in 60% isopropanol) for 15 min. The sections were then rapidly incubated in 60% isopropanol for several seconds and rinsed with distilled water. Cell nuclei could be counterstained with DAPI if necessary. After mounting, the sections were observed for red lipid deposition areas under a light or fluorescence microscope, and lipid area was quantified using ImageJ software.

[0160] The results are as follows Figure 17-18 As shown, Figure 17 The comparison of Oil Red staining results of brain tissues of mice in different treatment groups. Figure 18 The comparison results of Oil Red staining of brain tissues of mice in different treatment groups. Oil Red O staining showed that lipid droplets were obviously deposited in the brain tissue of the PBS group, slightly improved in the Exo and Lip groups, and significantly reduced in the Exo-Lip group.

[0161] 3. qRT-PCR analysis

[0162] In addition, qRT-PCR analysis was used to examine the expression of lipid metabolism-related genes, Dagt, CD36, and Pnlip, induced by MCAO / R. The specific method was as follows: after mice were sacrificed, the brain (ischemic hemisphere) was quickly removed and rinsed with PBS. Total RNA was extracted using Trizol reagent, and the concentration and purity were determined before reverse transcription into cDNA. qRT-PCR reactions were performed using the SYBR Green dye system on a real-time fluorescence quantitative PCR instrument. Three replicate wells were set up for each sample, and GAPDH was used as an internal reference gene. Relative expression levels were calculated using the 2^–ΔΔCt method, and the results were used to analyze the expression changes of each gene in the different treatment groups.

[0163] The results are as follows Figure 19 As shown, Figure 19 Figure 5 represents the expression levels of lipid metabolism-related genes in the brain tissues of mice in the different treatment groups. Figures A and B represent the expression levels of Dagt, CD36, and Pnlip, respectively. The results show that Dagt, CD36, and Pnlip were all upregulated, suggesting a role in lipid accumulation. Furthermore, combined Exo-Lip treatment significantly downregulated the expression of these genes, particularly Pnlip, demonstrating its superiority in regulating lipid metabolism genes.

[0164] Effect Experiment Example 2: Safety Evaluation of Exo-Lip

[0165] In order to systematically evaluate the safety of Exo-Lip in vivo, its biocompatibility was comprehensively investigated through histological observation and analysis of blood and serum biochemical indicators.

[0166] 1. Changes in tissue morphology

[0167] For each group of mice treated with Exo-Lip, samples were collected 24 hours after injection, and major organs such as the heart, liver, spleen, lungs and kidneys were collected for H&E staining to evaluate tissue morphological changes.

[0168] The results are as follows Figure 20 As shown, Figure 20 The following is a comparison of HE staining results of the heart, liver, spleen, lung, and kidney tissues of mice in different treatment groups. The results show that compared with the PBS control group, the tissue structures of the major organs in the Exo-Lip treatment group were intact, and no toxic manifestations such as cell swelling, congestion, edema, tissue necrosis, or obvious inflammatory cell infiltration were observed, indicating its good tissue compatibility.

[0169] 2. Routine peripheral blood test

[0170] Its systemic toxicity was assessed by routine peripheral blood tests, and its peripheral blood routine indicators were detected.

[0171] The results are as follows Figure 21 As shown, Figure 21 are the blood routine indicators of mice in different treatment groups, among which AH represent red blood cell count (RBC), white blood cell count (WBC), platelet count (PLT) and hemoglobin (HGB) level, liver function indicators alanine aminotransferase (ALT), aspartate aminotransferase (AST), and renal function indicators blood urea nitrogen (BUN) and creatine kinase (CK) levels.

[0172] Hematological analysis showed that the red blood cell count (RBC), white blood cell count (WBC), platelet count (PLT), and hemoglobin (HGB) levels of the Exo-Lip group were not significantly different from those of the PBS control group, suggesting that Exo-Lip had no significant adverse effects on the hematopoietic system. Furthermore, serum biochemical analysis revealed that the liver function markers alanine aminotransferase (ALT) and aspartate aminotransferase (AST), as well as the renal function markers blood urea nitrogen (BUN) and creatine kinase (CK), were not significantly different between the groups, further confirming that Exo-Lip had no significant effect on the functions of important metabolic organs such as the liver and kidneys.

[0173] In summary, multi-dimensional analyses of histology, hematology, and serum biochemistry all demonstrated that Exo-Lip exhibited excellent biocompatibility and low toxicity in mice and had good safety, laying a solid foundation for its further application in the treatment of ischemic brain injury.

[0174] Effect Experiment Example 3: Exploring the Molecular Mechanism Mediated by Exo-Lip

[0175] To further explore the potential molecular mechanisms of Exo-Lip in treating ischemic brain injury, transcriptomic analysis was performed on brain tissue from mice treated with Exo-Lip and PBS controls. Mice were sacrificed 24 hours after reperfusion, and brain tissue from the ischemic hemisphere was rapidly harvested and snap-frozen in liquid nitrogen. Eukaryotic mRNA with a poly(A) tail was enriched using Oligo(dT)-labeled magnetic beads, and then sheared using ultrasound. First-strand cDNA was synthesized using the fragmented mRNA as a template and random oligonucleotides as primers using the M-MuLV reverse transcriptase system. RNA was then degraded with RNase H, and second-strand cDNA was synthesized using dNTPs using the DNA polymerase I system. Purified double-stranded cDNA was end-repaired, A-tailed, and ligated with sequencing adapters. Approximately 200 bp of cDNA was selected using AMPure XP beads, amplified by PCR, and the PCR product was purified again using AMPure XP beads to generate a library. Qualified RNA samples were then used for library construction and sequencing.

[0176] The library was constructed using poly A tailing to enrich mRNA, and high-throughput paired-end sequencing (150bp) was performed on the Illumina platform. After sequencing, the raw data was filtered to remove adapter sequences and low-quality reads. After alignment to a reference genome (e.g., GRCm38), transcript quantification was performed using HTSeq or featureCounts. Expression levels were expressed as FPKM or TPM.

[0177] Differential expression analysis was performed using the edgeR software package, with a screening criteria of |log2FoldChange|≥1 and p<0.05. GO functional enrichment analysis and KEGG pathway enrichment analysis were then performed to clarify the enrichment characteristics of differentially expressed genes in biological processes and signaling pathways.

[0178] The results are as follows Figure 22-25 As shown, Figure 22 This is a heat map of RNA sequencing results for different groups of mice. Figure 23 A volcano plot of RNA sequencing results from different groups of mice was shown. The results showed that a total of 716 differentially expressed genes (DEGs) were screened, including 524 upregulated genes and 192 downregulated genes. The heat map and volcano plot visually demonstrate the significant transcriptional expression changes induced by Exo-Lip treatment.

[0179] Figure 24 The KEGG pathway enrichment analysis revealed that multiple metabolism and signal transduction pathways were significantly enriched in the DEGs. Among them, fat digestion and absorption, neuroactive ligand-receptor interaction, ether lipid metabolism, and JAK-STAT signaling pathways were particularly noteworthy due to their key roles in regulating neuroprotection, membrane homeostasis, and inflammatory responses.

[0180] Figure 25Gene set enrichment analysis revealed significant pathways, with A to D representing the fat digestion and absorption pathway, the neuroactive ligand-receptor interaction pathway, the ether lipid metabolism pathway, and the JAK-STAT signaling pathway, respectively. Gene set enrichment analysis (GSEA) revealed that, although some pathways did not reach traditional statistical significance thresholds, their enrichment trends were still biologically significant. The fat digestion and absorption pathway exhibited a positive normalized enrichment score (NES = 0.9551) in the Exo-Lip group, suggesting upregulation of related genes, potentially contributing to energy metabolism and lipid homeostasis. The neuroactive ligand-receptor interaction pathway (NES = 1.0369) also showed an upregulation trend, suggesting that Exo-Lip may regulate interneuronal signaling and synaptic activity. The significant enrichment of the ether lipid metabolism pathway (NES = 1.3706) may be closely related to membrane lipid remodeling and the alleviation of oxidative stress. The JAK-STAT signaling pathway showed negative enrichment (NES = –1.003), suggesting that this pathway may be inhibited, thereby attenuating proinflammatory responses and abnormal cell proliferation, which is consistent with the previously observed anti-inflammatory effects.

[0181] Transcriptomic analysis revealed that Exo-Lip may exert its neuroprotective effects by synergistically regulating key pathways such as lipid metabolism, neural signaling, and inflammatory responses. These molecular mechanisms provide a solid theoretical basis and biological support for its therapeutic potential in central nervous system (CNS) injury.

Claims

1. A method for preparing exosome-liposome composite nanoparticles, characterized in that: The method comprises the following steps: fusing the exosomes derived from neural stem cells with the liposomes encapsulating Yulangsan polysaccharide to obtain the product.

2. The method for preparing exosome-liposome composite nanoparticles according to claim 1, wherein: The membrane fusion is performed by reverse phase evaporation method, extrusion method, freeze-thaw method, ultrasonic incubation method, electric fusion method or microfluidics method.

3. The method for preparing exosome-liposome composite nanoparticles according to claim 2, wherein: The ultrasonic incubation method was used to perform fusion according to the following steps: exosomes and liposomes were taken, placed in a PBS solution, and incubated after ultrasonication.

4. The method for preparing exosome-liposome composite nanoparticles according to claim 3, wherein: Meet at least one of the following conditions: (1) The mass ratio of the exosomes to the liposomes is 3:1-1:3, for example, 3:1, 2:1, 1:1, 1:2, 1:3, preferably 1:1; (2) The concentration of the PBS solution is 0.01-0.02M, and the pH is 7.2-7.

6. Preferably, the concentration is 0.01M, and the pH is 7.4 (3) The concentration of the exosomes in the PBS solution is 0.01-0.2 μg / μL, preferably 0.2 μg / μL; (4) The power of the ultrasound is 50-300W, preferably 80W; (5) The ultrasonic time is 5-20 minutes, preferably 10 minutes; (6) The ultrasound is performed in a cycle of 2-5 seconds on and 5-8 seconds off, preferably in a cycle of 3 seconds on and 7 seconds off; (7) The incubation temperature is 4-42°C, preferably 37°C; (8) The incubation time is 0.5-2 hours, preferably 1 hour.

5. The method for preparing exosome-liposome composite nanoparticles according to claim 1, wherein: The neural stem cell-derived exosomes are prepared by the following method: performing cell culture on neural stem cells, collecting the culture supernatant, and obtaining exosomes by centrifugation; Preferably, the method for preparing the exosomes meets at least one of the following conditions: (1) The neural stem cells are obtained by the following method: peripheral blood mononuclear cells are taken, and Sendai virus containing transcription factors Oct4, Sox2, Klf4 and c-Myc is added for transfection, and then peripheral blood mononuclear cell expansion medium is added for culture, the cells are collected, hiNPCs medium is added, and the cells are inoculated on a plate covered with poly-D-lysine hydrobromide and laminin, and the cells are centrifuged and then cultured until hiNPCs clones appear, thereby obtaining neural stem cells; (2) The method for collecting the culture supernatant is as follows: culturing neural stem cells in serum-free medium, discarding the old medium and replacing it with fresh medium when the cell density reaches 70% to 80%, culturing the cells in a cell culture incubator for 48 ± 12 hours, and then collecting the culture supernatant; (3) The centrifugation conditions are as follows: centrifugation at 300±100×g and 4±2°C for 20±5 min; taking the supernatant and centrifuging at 2000±100×g and 4±2°C for 20±5 min; taking the supernatant and filtering it through a 0.22 μm filter membrane, and continuing to centrifuge at 10,000±100×g and 4±2°C for 1±0.1 h; taking the supernatant and centrifuging it at 100,000±1000×g and 4±2°C for 90±5 min, and discarding the supernatant to obtain the product; preferably, centrifugation at 300×g and 4°C for 20 min; taking the supernatant and centrifuging it at 2000×g and 4°C for 20 min; taking the supernatant and filtering it through a 0.22 μm filter membrane, and continuing to centrifuge it at 10,000×g and 4°C for 1 h; taking the supernatant and centrifuging it at 100,000×g and 4°C for 90 min, and discarding the supernatant to obtain the product.

6. The method for preparing exosome-liposome composite nanoparticles according to claim 1, wherein: The liposomes encapsulating the Yulangsan polysaccharide are prepared by the following method: soybean lecithin, cholesterol and Tween 80 are dissolved in a first organic solvent, the organic solvent is removed by rotary evaporation to form a lipid film layer on the inner wall of a container, a second organic solvent and a PBS solution of the Yulangsan polysaccharide are added simultaneously, ultrasonication is performed to form a uniform dispersion, the solvent is then evaporated by rotary evaporation to form a milky white or translucent dispersion, a PBS dispersion solution is added, rotary evaporation is continued until the solution becomes an aqueous suspension, ultrasonication is performed to further disperse the system, and filtration is performed to remove macromolecules, thereby obtaining the liposomes; Preferably, the method for preparing the liposomes meets at least one of the following conditions: (1) The mass ratio of soybean lecithin, cholesterol and Tween 80 is 80-90:20-10:10-5, preferably 85:15:8.5; (2) The first organic solvent is chloroform; (3) The second organic solvent is diethyl ether; (4) The volume ratio of the second organic solvent to the PBS solution of the Yulangsan polysaccharide is 10-2:1, preferably 5:1; (5) The ratio of the soybean lecithin to the second organic solvent is 5-15 mg:1 mL, preferably 10.2 mg:1 mL; (6) The concentration of Yulangsan polysaccharide in the PBS solution of Yulangsan polysaccharide is 1-10 mg / mL, preferably 5 mg / mL; (7) The time taken for ultrasonication to form a uniform dispersion is 10-60 min, preferably 30 min; (8) Ultrasonication is used to further disperse the system for 5-30 minutes, preferably 10 minutes, and the process is repeated in a cycle of 3 seconds on and 7 seconds off. (9) The pore size of the filter membrane used to filter out macromolecules is 0.22-0.45 μm, preferably 0.22 μm; (10) The obtained liposomes are stored at 4-37°C, preferably at 4°C.

7. The method for preparing exosome-liposome composite nanoparticles according to claim 1, wherein: The Yulangsan polysaccharide is prepared by the following method: taking Yulangsan, adding ethanol and water solution to extract, adding water to the residue after the extract is concentrated, concentrating the water extract into an extract, adding ethanol, standing and precipitating, and taking the precipitate to obtain Yulangsan polysaccharide; Preferably, the method for preparing the Yulangsan polysaccharide meets at least one of the following conditions: (1) The volume percentage concentration of the ethanol aqueous solution is 50-80%, preferably 70%; (2) The usage ratio of the Yulangsan and the ethanol aqueous solution is 1 kg:5-15 L, for example, 1 kg:10 L; (3) The extraction with the ethanol aqueous solution is reflux extraction, and the extraction time is 1-5 hours, preferably 3 hours; (4) The ratio of the amount of the Yulang umbrella and the water added to the residue is 1 kg:5-15 L, for example, 1 kg:10 L; (5) The residue is extracted by boiling in water for 1-5 hours, preferably 3 hours; the number of extractions is 2-4 times, preferably 3 times; (6) adding ethanol after the water extract is concentrated to a specific gravity of 1.0-1.2 at 50° C., preferably adding ethanol after the water extract is concentrated to a specific gravity of 1.1 at 50° C.; (7) After the water extract is concentrated, the volume of ethanol added is 2-4 times that of the extract, preferably 3 times; (8) The precipitate is further vacuum-dried at 60-80° C. to obtain powdered Yulangsan polysaccharide.

8. Exosome-liposome composite nanoparticles prepared by the preparation method according to any one of claims 1 to 7.

9. The exosome-liposome composite nanoparticles according to claim 8, wherein Meet at least one of the following conditions: (1) The particle size of the composite nanoparticles is 100-200 nm, preferably 170-200 nm, for example 190±5 nm; (2) The exosomes are exosomes of neural stem cells obtained by inducing differentiation of PBMCs.

10. Use of the exosome-liposome composite nanoparticles according to any one of claims 8 to 9 in the preparation of a drug for preventing and / or treating ischemic stroke; Preferably, the composite nanoparticles exert their effects by protecting and repairing nerves, promoting recovery of cerebral blood flow, and / or reducing infarct volume.

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