Targeted external vesicle as well as preparation method and application thereof

Abstract

The invention discloses a targeted external vesicle as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The targeted outer vesicle comprises a Yulangsan outer vesicle and a cerebral ischemia homing peptide, wherein the Yulangsan outer vesicle and the cerebral ischemia homing peptide are directly coupled or coupled through a phospholipid-PEG (Polyethylene Glycol) conjugate. The targeted external vesicle is prepared by the following steps: firstly, extracting and obtaining an external vesicle with anti-inflammatory, anti-oxidative damage and anti-apoptosis effects from plant Yulangsan, and coupling the external vesicle with SHp peptide to prepare an engineered plant external vesicle with targeting capability, namely the targeted external vesicle. The targeted external vesicle endows the Yulangsan external vesicle with the ability of targeting cerebral ischemic tissues, and improves the effect of treating ischemic cerebral apoplexy.

Description

Technical Field

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

[0002] Ischemic stroke, defined as cerebral vascular stenosis or occlusion, leads to brain tissue loss of blood, oxygen, and necrosis. It has become the second leading cause of death worldwide. Current research shows that most drugs have difficulty crossing the blood-brain barrier (BBB), posing a major challenge in treating brain diseases. Furthermore, even after crossing the BBB, drugs often lack targeting.

[0003] Therefore, it is of great research significance to develop effective drugs that can break through the BBB and target ischemic stroke. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the defect in the prior art of lacking effective drugs that can break through the BBB and target ischemic stroke, and to provide a targeted exosome that can break through the BBB and achieve precise delivery, thereby effectively improving ischemic stroke.

[0005] The present invention solves the above technical problems through the following technical solutions: a targeted extracellular vesicle, comprising Yulangsan extracellular vesicles and cerebral ischemia homing peptide, wherein the Yulangsan extracellular vesicles and the cerebral ischemia homing peptide are directly coupled or coupled through a phospholipid-PEG conjugate.

[0006] The inventors believe that plant-derived exosome-like nanovesicles (EVs) have the potential for therapeutic applications due to their low immunogenicity, low cost, and ease of large-scale production compared to traditional animal-derived or cell-culture-derived exosomes. EVs also leverage their small size (30-150 nm), lipid bilayer structure, and surface molecules to facilitate cross-sectional transport across the BBB. YLS (Millettia pulchra (Benth.) Kurz var. laxior (Dunn) Z. Wei), a herbal remedy used by the Guangxi Zhuang ethnic group, has been shown to enhance intelligence, fight aging, and enhance immunity. Previous studies have revealed that YLS polysaccharide (YLSP) exhibits anti-inflammatory and antioxidant properties, leading the inventors to consider whether YLS-derived EVs could improve ischemic stroke. However, preliminary experiments have shown that YLS-derived EVs lack targeting, reducing both target concentration and efficacy, necessitating structural modification.

[0007] Based on this research, the inventors first extracted and obtained exovesicles from the plant Yulangsan, which possess anti-inflammatory, antioxidant, and anti-apoptotic properties. They then coupled these exovesicles with SHp peptides to create engineered plant exovesicles with targeting capabilities. This method imbued Yulangsan exovesicles with the ability to target ischemic brain tissue, enhancing their effectiveness in treating ischemic stroke.

[0008] In one embodiment, the Yulangsan extracellular vesicles and the cerebral ischemia homing peptide are coupled via Mal-PEG-DSPE. Preferably, the number average molecular weight of the Mal-PEG-DSPE is 1900-2100 Da, for example, 2000 Da. Mal-PEG-DSPE within this molecular weight range enables better coupling with the Yulangsan extracellular vesicles.

[0009] In one embodiment, the Yulangsan extracellular vesicles are prepared by the following method:

[0010] Take Yulangsan, add PBS solution to homogenize, coarse filter, take the filtrate and ultracentrifuge to remove cell debris, take the filtrate and high-speed centrifuge again to obtain a precipitate, which is the Yulangsan extracellular vesicle.

[0011] In one embodiment, the Yulangsan is fresh, undried Yulangsan. Using fresh plants, due to their high cellular activity, results in relatively stable exovesicle release and content, reducing exovesicle changes and impurity buildup caused by cell death or degradation, thereby improving extraction efficiency and the purity and integrity of the exovesicles.

[0012] In one embodiment, the PBS solution is 0.01±0.005 M, pH=7.2-7.4.

[0013] In one embodiment, the PBS solution is pre-cooled to 2-6°C before homogenization, preferably 4°C.

[0014] In one embodiment, the coarse filtration is performed by filtering with gauze.

[0015] In one embodiment, the ultracentrifugation and high-speed centrifugation are both performed at 4-25°C, preferably 4°C.

[0016] In one embodiment, the ultracentrifugation is performed according to the following program: 500±200×g for 10±5 min, 3,000±1,000×g for 30±10 min, 10,000±2,000×g for 50±10 min, and 40,000±10,000×g for 40±10 min.

[0017] In one embodiment, the ultracentrifugation is performed according to the following procedure: 500×g for 10 min, 3,000×g for 30 min, 10,000×g for 50 min, and 40,000×g for 40 min. High-quality Yulangsan extracellular vesicles can be obtained by centrifugation under these conditions.

[0018] In one embodiment, the high-speed centrifugation condition is: 130,000±30,000×g for 80±20 min; preferably 130,000×g for 80 min.

[0019] In one embodiment, the precipitate is obtained by high-speed centrifugation, resuspended in PBS solution, and stored at -80°C.

[0020] The present invention also discloses a method for preparing targeted exosomes, comprising the following steps: taking Yulangsan exosomes, and directly coupling the Yulangsan exosomes with cerebral ischemia homing peptides or coupling them through a phospholipid-PEG conjugate by using a click chemistry method, a carbodiimide method or a lipid insertion method.

[0021] In one embodiment, the Yulangsan extracellular vesicles are prepared using the above-mentioned method for preparing Yulangsan extracellular vesicles.

[0022] In one embodiment, the Yulangsan extracellular vesicles and the cerebral ischemia homing peptide are coupled by click chemistry, and the specific method is as follows: take the Yulangsan extracellular vesicle solution, add dibenzocyclooctylthio-N-hydroxysuccinimide ester solution, mix, react, so that the surface of the Yulangsan extracellular vesicles is modified with dibenzocyclooctyne groups, and then drop the azide-modified cerebral ischemia homing peptide to react and couple.

[0023] In one embodiment described above, the azide-modified cerebral ischemia homing peptide is prepared by the following method: dissolving the SHp peptide in PBS and then transferring it to a sodium borate buffer solution, adding azide-polyethylene glycol-N-hydroxysuccinimide ester, adding glycine after the reaction to terminate the reaction, adding acetone to the reaction system, centrifuging, and collecting the precipitate to obtain the azide-modified cerebral ischemia homing peptide.

[0024] In one embodiment, the Yulangsan extracellular vesicles and the cerebral ischemia homing peptide are coupled by the carbodiimide method. The specific method is as follows: take the Yulangsan extracellular vesicle solution, add carbodiimide and N-hydroxysuccinimide, mix, react to activate the carboxyl groups on the surface of the Yulangsan extracellular vesicles, and then drip the cerebral ischemia homing peptide into the solution for reaction coupling.

[0025] In one embodiment, the Yulangsan extracellular vesicles and the cerebral ischemia homing peptide are coupled via Mal-PEG-DSPE, specifically by the following method:

[0026] Preparation of SHp-PEG-DSPE: Mal-PEG-DSPE is dissolved in an organic solvent to obtain solution A, and brain ischemia homing peptide is dissolved in PBS to obtain solution B. Solutions A and B are mixed and reacted to covalently link the brain ischemia homing peptide to the maleimide group of Mal-PEG-DSPE via the thiol group of cysteine ​​to obtain SHp-PEG-DSPE;

[0027] Preparation of SHp-EVs: Take the Yulangsan extracellular vesicle solution, add the SHp-PEG-DSPE, and carry out coupling reaction to obtain SHp-EVs.

[0028] In one embodiment, the Yulangsan extracellular vesicles and the cerebral ischemia homing peptide are coupled via Mal-PEG-DSPE, and in the step of preparing SHp-PEG-DSPE, the number average molecular weight of the Mal-PEG-DSPE is 1900-2100 Da, for example, 2000 Da.

[0029] In one embodiment, in the step of preparing SHp-PEG-DSPE, the concentration of Mal-PEG-DSPE in solution A is 3.8-4.1 mg / mL, for example, 4 mg / mL.

[0030] In one embodiment, in the step of preparing SHp-PEG-DSPE, the concentration of cerebral ischemia homing peptide in the solution B is 10-12 mg / mL, for example, 10 mg / mL.

[0031] In one embodiment, in the step of preparing SHp-PEG-DSPE, the mass ratio of Mal-PEG-DSPE to cerebral ischemia homing peptide is 1:1.5-1:2.8, for example, 1:2.5.

[0032] In one embodiment, in the step of preparing SHp-PEG-DSPE, the organic solvent is N,N-dimethylformamide.

[0033] In one embodiment, in the step of preparing SHp-PEG-DSPE, the PBS is a 0.01±0.005M PBS solution with a pH of 7.4.

[0034] In one embodiment, in the step of preparing SHp-PEG-DSPE, the reaction is carried out under the protection of an inert gas, such as nitrogen.

[0035] In one embodiment, in the step of preparing SHp-PEG-DSPE, the reaction is carried out at 10-40°C, for example, 20-30°C.

[0036] In one embodiment, in the step of preparing SHp-PEG-DSPE, the reaction is carried out under light-proof conditions.

[0037] In one embodiment, in the step of preparing SHp-PEG-DSPE, the reaction time is 4-12 hours, for example, 8 hours.

[0038] In one embodiment, after the step of preparing SHp-PEG-DSPE, a purification step is further included, wherein the purification step comprises dialyzing SHp-PEG-DSPE against water and freeze-drying for storage. Preferably, the dialysis uses a dialysis membrane with a molecular weight cutoff of 3.5 kDa.

[0039] In one embodiment, in the step of preparing SHp-EVs, the concentration of the Yulangsan extracellular vesicles is 0.1-1 mg / mL, for example, 0.5 mg / mL.

[0040] In one embodiment, in the step of preparing SHp-EVs, the reaction concentration of SHp-PEG-DSPE is 0.1-25 μM, preferably 1-5 μM, and more preferably 5 μM.

[0041] In one embodiment, in the step of preparing SHp-EVs, the dosage ratio of the Yulangsan extracellular vesicles to the SHp-PEG-DSPE is 0.5 mg / mL:0.1-25 μM, preferably 0.5 mg / mL:1-5 μM, and more preferably 0.5 mg / mL:5 μM.

[0042] In one embodiment, in the step of preparing SHp-EVs, the temperature of the coupling reaction is 4-10°C, for example, 4°C.

[0043] In one embodiment, in the step of preparing SHp-EVs, the coupling reaction time is 12-24 hours.

[0044] In one embodiment, after the step of preparing SHp-EVs, a removal step is further included, wherein the unreacted raw materials are removed from the coupling reaction product by ultrafiltration; preferably, the ultrafiltration method is to use an ultrafiltration tube with a molecular weight cutoff of 100 kDa, centrifuge at 14,000 × g for 10 minutes, and repeat 3 times.

[0045] On the other hand, the present invention also discloses the targeted extracellular vesicles prepared by the above preparation method.

[0046] The present invention also discloses the use of the above-mentioned targeted extracellular vesicles in the preparation of a medicine for improving cerebral ischemia.

[0047] In one embodiment, the drug for improving cerebral ischemia is used to treat ischemic stroke.

[0048] In one embodiment, the targeted extracellular vesicles improve cerebral ischemia symptoms by reducing inflammatory factors, apoptotic factors and / or phosphorylated proteins in brain tissue and increasing anti-apoptotic factors.

[0049] In one embodiment, the inflammatory factor is TNF-α, IL-6 and / or IL-1β; the apoptotic factor is Bax, the anti-apoptotic factor is Bcl-2, and the phosphorylated proteins are MEKK1, JNK and P38.

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

[0051] The targeted exosomes of the present invention are first extracted from the plant Yulangsan to obtain exosomes with anti-inflammatory, antioxidant, and anti-apoptotic effects. These exosomes are then coupled with SHp peptide to create engineered plant exosomes with targeting capabilities. These targeted exosomes are endowed with the ability to target cerebral ischemic tissue, enhancing the effectiveness of treating ischemic stroke. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 The yields of SHp-EVs prepared by different coupling methods.

[0053] Figure 2 The yields of SHp-EVs prepared with different concentrations of SHp-PEG-DSPE.

[0054] Figure 3 Transmission electron microscopy images of extracellular vesicles, where A and B are EVs and SHp-EVs, respectively.

[0055] Figure 4 Representative immunoblot bands of exosome markers in EVs and SHp-EVs.

[0056] Figure 5 Colocalization fluorescence images, where A, B, and C are FITC-SHp-PEG-DSPE (green), DiR-labeled EVs (red), and colocalization (yellow), respectively.

[0057] Figure 6 The ELISA method was used to detect the content of verification factors in LPS inflammatory cells, where A is NO, B is TNF-α, C is IL-6, and D is IL-1β.

[0058] Figure 7 Images of DIO-EVs and DIO-SHp-EVs uptake by HT22 cells after OGD / R reoxygenation and resugarization for 3 h.

[0059] Figure 8 The in vivo distribution of SHp-EVs was imaged by coupling different concentrations of SHp-PEG-DSPE to EVs.

[0060] Figure 9 Brain fluorescence quantification of SHp-EVs obtained by coupling different concentrations of SHp-PEG-DSPE with EVs.

[0061] Figure 10 Brain fluorescence imaging of the distribution of EVs and SHp-EVs in vivo.

[0062] Figure 11 Brain fluorescence quantification of the distribution of EVs and SHp-EVs in vivo.

[0063] Figure 12 Fluorescence imaging of EVs and SHp-EVs in brain tissue.

[0064] Figure 13 Fluorescence quantification of EVs and SHp-EVs in brain tissue.

[0065] Figure 14 This is a TTC staining image of brain tissue.

[0066] Figure 15 The infarct volume was calculated by TTC staining of brain tissue.

[0067] Figure 16 This is a gait footprint diagram.

[0068] Figure 17 This is the motion trajectory diagram of the open field experiment.

[0069] Figure 18 This is a statistical chart of the total movement distance, movement distance in the central area, percentage of movement distance in the central area, and percentage of time in the central area of ​​the mice in the open field test.

[0070] Figure 19 The ELISA method was used to measure the levels of inflammatory factors in brain tissue, where A, B, and C were TNF-α, IL-6, and IL-1β, respectively.

[0071] Figure 20 Representative cytokines detected by qPCR in RNA extracted from brain tissue.

[0072] Figure 21 Representative protein blot bands.

[0073] Figure 22 for Figure 21 The expression levels of each protein in .

[0074] Figure 23The images are hematoxylin-eosin stained.

[0075] Figure 24 Nissl-stained images. DETAILED DESCRIPTION

[0076] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples.

[0077] Unless otherwise specified, the reagents used in the following examples are all commercially available. The methods used in the following examples are all conventional methods unless otherwise specified.

[0078] The following examples use PBS, which is a 0.01 M phosphate buffer solution with a pH of 7.2-7.4.

[0079] Yulangsan polysaccharide (YLSP) was prepared according to the following method:

[0080] 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.

[0081] Example 1

[0082] Extraction of extracellular vesicles (EVs) from Yulangsan.

[0083] Fresh Yulangsan plants were ground and extracted in PBS (0.01M, pH 7.2-7.4) at 4°C at a material-liquid ratio of 1:5 (5 mL of extraction solution was added per gram of raw material). High-speed homogenization was repeated 10 times using an electric blender (15,000 rpm), each for 15 seconds, and the mixture was filtered through gauze. The filtrate was collected and ultracentrifuged at 4°C to remove large particles and cell debris. Ultracentrifugation conditions were: 500 × g for 10 minutes, 3000 × g for 30 minutes, 10,000 × g for 50 minutes, and 40,000 × g for 40 minutes, retaining the supernatant. Finally, the supernatant was discarded after centrifugation at 130,000 × g for 80 minutes to obtain Yulangsan extracellular vesicles (EVs), which were resuspended in PBS and stored at -80°C until further use.

[0084] Example 2

[0085] Preparation of Yulangsan-targeted extracellular vesicles.

[0086] After preliminary investigation and screening, we chose to couple brain ischemia homing peptide (SHp) with Yulangsan extracellular vesicles (EVs), and compared different coupling methods.

[0087] 1. Experimental Methods

[0088] 1. Click Chemistry

[0089] The EVs solution was diluted to 0.5 mg / mL with PBS as a solvent, and dibenzocyclooctylthio-N-hydroxysuccinimide ester solution (DBCO-NHS, final concentration of 3 μM) was added. The mixed solution was reacted on a rotating mixer at room temperature for 2 h to allow the NHS ester group of DBCO-NHS to react with the amino group on the EVs surface, thereby modifying the EVs surface with DBCO groups (dibenzocyclooctyne groups).

[0090] The SHp peptide (SEQ ID NO: 1, purchased from Shanghai Jier Biochemical Co., Ltd.) was dissolved in PBS and transferred to sodium borate buffer (0.1 M, pH 8.3). Azide-polyethylene glycol-N-hydroxysuccinimide ester (N3-PEG-NHS, molecular weight 5000 Da, purchased from Merck) was added at a molar ratio of 1:1.5. The mixture was reacted at 4°C on a rotating mixer in the dark for 2 h. The reaction was terminated by the addition of glycine solution (final concentration 50 mM, prepared in PBS). The resulting reaction solution was incubated with acetone at -20°C for 1 h and then centrifuged at 12,000 rpm for 10 h. The precipitate was collected and the unmodified peptide was dissolved in 20% acetonitrile / water with shaking. The precipitate was then centrifuged again to obtain the azide-modified SHp ​​peptide.

[0091] Then, the azide-modified SHp ​​peptide solution (final concentration of 10 μM) was dropped into the EVs solution with DBCO groups on the surface, and the reaction was carried out on a rotating mixer at 4°C overnight to allow DBCO-EVs to undergo click chemistry reaction with the azide groups, thereby achieving coupling of the peptide and EVs.

[0092] To remove excess DBCO-NHS and unbound peptides, the SHp-EVs were obtained by centrifugation at 14,000 × g for 10 min using an ultrafiltration tube (molecular weight cutoff 100 kDa) and washed three times with PBS.

[0093] 2. Carbodiimide method

[0094] The EVs solution was diluted to 0.5 mg / mL with PBS as solvent, and carbodiimide (EDC, final concentration of 2 mM) and N-hydroxysuccinimide (NHS, final concentration of 5 mM) were added. The mixed solution was reacted on a rotating mixer at room temperature for 2 h to activate the carboxyl groups on the EVs surface.

[0095] Then, SHp peptide solution (final concentration of 10 μM, sequence CLEVSRKNC, SEQ ID NO: 1, purchased from Shanghai Jier Biochemical Co., Ltd.) was added dropwise, and the pH value was adjusted to 7-8 with Tris-HCl buffer (pH = 7.4). The mixture was reacted on a rotating mixer at 4°C overnight to allow the amino group of the SHp peptide to undergo a condensation reaction with the activated carboxyl group of EVs to form an amide bond, thereby achieving coupling between the two.

[0096] After the reaction, glycine solution (final concentration 50 mM, prepared in PBS) was added to terminate the reaction. To remove excess cross-linker and unbound peptide, the SHp-EVs were obtained by centrifugation at 14,000 × g for 10 min using an ultrafiltration tube (molecular weight cutoff 100 kDa) and washed three times with PBS.

[0097] 3. Lipid Insertion Method

[0098] Mal-PEG-DSPE (MW = 2000Da, 4mg, purchased from Xibao Biotechnology Co., Ltd.) and SHp ​​peptide (10mg, sequence CLEVSRKNC, purchased from Shanghai Jier Biochemical Co., Ltd.) were dissolved in 1mL N,N-dimethylformamide (DMF) and 10mL PBS (0.01±0.005M, pH 7.4), respectively. The two were mixed and reacted under nitrogen protection, and SHp ​​was covalently linked to the maleimide group of Mal-PEG-DSPE via the thiol group of cysteine.

[0099] The product was then dialyzed against distilled water (dialysis membrane with a molecular weight cutoff of 3.5 kDa) and freeze-dried to obtain SHp-PEG-DSPE. SHp-PEG-DSPE was detected by mass spectrometry, and the molecular weight of the product was in the range of 3700-3900 Da.

[0100] The EVs solution was diluted to 0.5 mg / mL with PBS, SHp-PEG-DSPE (final concentration of 10 μM) was added, and the mixture was reacted on a rotating mixer at 4°C overnight.

[0101] After the reaction, in order to remove excess unbound SHp-PEG-DSPE, ultrafiltration tubes (molecular weight cutoff = 100 kDa) were used for centrifugation at 14,000 × g for 10 min and washed three times with PBS to obtain SHp-EVs.

[0102] 2. Experimental Results

[0103] The binding yield of SHp-EVs obtained by each method was calculated, and the protein concentration was detected using a BCA kit (purchased from Shanghai Biyuntian Biotechnology Co., Ltd.) to evaluate the yield of SHp-EVs obtained by different coupling methods. The specific method is as follows:

[0104] The SHp-EVs obtained after ultrafiltration were added to PBS and diluted to a volume of 100 μL. 20 μL of the diluted sample was taken and added to 200 μL of BCA working solution. After incubation in a 37°C incubator for 30 minutes, the absorbance at a wavelength of 562 nm was measured using a microplate reader. According to the absorbance value, the corresponding protein concentration was calculated by substituting the standard curve, and then multiplied by the dilution factor to obtain the protein concentration of the sample. At the same time, the protein concentration of the sample before ultrafiltration (100 μL) was determined in the same way. Binding yield = sample protein concentration before ultrafiltration / sample protein concentration after ultrafiltration × 100%

[0105] The results are as follows Figure 1 As shown, Figure 1 The binding yields of SHp-EVs were calculated for different coupling methods. The results showed that at a peptide reaction concentration of 10 μM, the yields of SHp-EVs using the click chemistry, carbodiimide, and lipid intercalation methods were 44.0%, 9.2%, and 48.1%, respectively. This indicates that the binding yield of the carbodiimide method was significantly lower than that of the click chemistry and lipid intercalation methods. The lipid intercalation method had the best binding yield, and considering all factors, the lipid intercalation method was selected as the method for preparing SHp-EVs in subsequent experiments.

[0106] Example 3

[0107] Optimization of the lipid insertion method.

[0108] To optimize the peptide concentration with the highest binding efficiency, the SHp-PEG-DSPE concentration gradient in the reaction system (0.1, 0.5, 1.0, 5.0, 10, and 25 μM) was set and mixed with the EV solution. The mixture was then reacted overnight at 4°C on a rotating mixer. Other conditions were the same as in Example 1.

[0109] The binding yield of SHp-EVs was determined as Figure 2 The figure shows the binding yield of SHp-EVs prepared with different concentrations of SHp-PEG-DSPE. The results show that when the SHp-PEG-DSPE concentration increases from 0.1μM to 1.0μM, the binding yield increases with the increase in SHp-PEG-DSPE concentration. However, as the SHp-PEG-DSPE concentration continues to increase, the product binding yield begins to decrease. The binding yields of SHp-PEG-DSP with EVs at 0.1, 0.5, 1, 5, 10, and 25μM are 63.1%, 67.0%, 85.5%, 54.2%, 48.1%, and 39.5%, respectively.

[0110] Example 4

[0111] The EVs obtained in Example 1 and the SHp-EVs obtained in Example 3 were selected as samples for characterization.

[0112] 1. Transmission electron microscopy characterization of samples

[0113] EVs and SHp-EVs (10 μL) were stained with uranyl acetate for 1 min and then dropped onto a 300-mesh carbon-coated copper grid. After natural drying at room temperature, the samples were observed using a transmission electron microscope. All images were taken at a voltage of 100 kV.

[0114] The results are as follows Figure 3 As shown, Figure 3 Transmission electron micrographs of EVs and SHp-EVs show that EVs are oval in shape, slightly concave in the interior, with smooth edges and a visible bilayer membrane. SHp-EVs are similar in shape to EVs, but have subtle protrusions on the membrane surface and less smooth edges. The average particle sizes of EVs and SHp-EVs are approximately 94 nm and 130 nm, respectively.

[0115] 2. Characteristic protein expression

[0116] Both EVs and SHp-EVs were lysed in RIPA buffer (containing protease inhibitors) on ice for 30 minutes. The cells were centrifuged at 12,000 × g for 15 minutes, and the supernatant was collected to obtain protein samples. Protein concentration was measured using a BCA assay. The sample loading volume (50 μg / well) was adjusted according to protein concentration. 5× loading buffer was added, mixed at a volume ratio of 1:4, and the proteins were denatured by boiling in a metal bath at 100°C for 5 minutes. Protein samples were separated by SDS-PAGE and transferred to PVDF membranes, blocked with 5% skim milk, and incubated with primary antibodies at a dilution of 1:1000 overnight at 4°C. The PVDF membranes were then incubated with secondary antibodies on a shaker at room temperature for 1 hour, and protein bands were detected using a gel imaging system.

[0117] The results are as follows Figure 4 As shown, Figure 4 The electrophoresis bands depicting the characteristic proteins of EVs and SHp-EVs show that both EVs and SHp-EVs have high expression of exosome characteristic proteins TSG101, CD81 and CD9.

[0118] 3. Fluorescence colocalization

[0119] The detection method is to stain SHp-PEG-DSPE with fluorescein isothiocyanate (FITC) and EVs with the lipophilic dye DIR at 37°C for 30 minutes, then centrifuge at 14,000g for 5 minutes using an ultrafiltration tube (100kDa). After removing excess dye, the obtained FITC-SHp-PEG-DSPE and DIR-EVs are incubated at 4°C for 12 hours, and the fluorescence of the samples is captured using a fluorescence microscope.

[0120] The results are as follows Figure 5 As shown, Figure 5 Figures A, B, and C are colocalization fluorescence images of FITC-SHp-PEG-DSPE (green), DIR-labeled EVs (red), and colocalization (Merge) images, respectively. The results show that FITC-labeled SHp-PEG-DSPE (green) and DIR-labeled EVs (red) colocalize (yellow) after overnight incubation, indicating that SHp-PEG-DSPE can be modified onto the surface of EVs via lipid insertion.

[0121] Example 5

[0122] Evaluation of the application of SHp-EVs in ischemic stroke

[0123] 1. Anti-inflammatory activity

[0124] 1. Method

[0125] To evaluate the anti-inflammatory activity of the samples in vitro, an inflammatory cell model was constructed using lipopolysaccharide (LPS).

[0126] HT22 cells (source: ATCC Cell Bank) were seeded in 48-well plates for 24 hours and then pretreated with YLSP, EVs, and SHp-EVs (50 μg / mL, 200 μL) for 24 hours. The drug-containing medium was discarded, and lipopolysaccharide (LPS, 1 μg / mL, 200 μL) was added to each well, except for the blank control group, and cultured for an additional 24 hours. The supernatant was aspirated and inflammatory cytokine levels were measured using ELISA kits (purchased from Fancovi).

[0127] 2. Results

[0128] The results are as follows Figure 6 As shown, Figure 6 The results of ELISA assays for LPS-induced inflammatory cell content are shown in Figure 1. A represents NO, B represents TNF-α, C represents IL-6, and D represents IL-1β. The results showed that compared with the LPS group, YLSP, EVs, and SHp-EVs all significantly reduced NO, TNF-α, IL-6, and IL-1β levels. The order of anti-inflammatory activity from highest to lowest was SHp-EVs > EVs > YLSP.

[0129] Targeted Uptake of SHp-EVs

[0130] 1. Method

[0131] To evaluate whether SHp-EVs can target apoptotic neurons, this example uses an in vitro glucose deprivation (OGD / R) cell model to simulate the ischemia-reperfusion injury process. Specifically, the mouse hippocampal neuronal cell line (HT22, source: ATCC cell bank) was plated in a 48-well plate for 24 hours, washed 3 times with PBS, replaced with a sugar-free medium, and placed in a three-gas incubator (37°C, 5% CO2, 95% N2) for 4 hours of hypoxia and glucose deprivation. The sugar-free medium was aspirated, and DIO-labeled EVs and SHp-EVs (50 μg / mL, 200 μL) prepared with high-glucose medium were added and placed in a 37°C, 5% CO2 constant temperature incubator for re-glucose and reoxygenation culture for 3 hours. The culture medium was aspirated, and the cells were washed twice with PBS. The cell nuclei were stained with Hoechst33342 solution (50 μM, purchased from Beijing Solebow Technology Co., Ltd.), incubated at 37°C for 15 min, and washed twice with PBS. Images of the cells taking up EVs and SHp-EVs were captured using an inverted fluorescence microscope.

[0132] The above-mentioned DIO-labeled EVs were prepared by the following method: EVs and SHp-EVs (protein concentration approximately 1 μg / μL) were incubated with DIO (1 mM) at a volume ratio of 500:1 in a dark chamber at 37°C for 30 min.

[0133] 2. Results

[0134] The results are as follows Figure 7 As shown, Figure 7 The results show that SHp-EVs are more enriched around apoptotic HT22 cells compared with EVs, indicating that SHp-EVs have a targeting effect on apoptotic neurons.

[0135] In vivo activity in middle cerebral artery occlusion (MCAO) mice

[0136] 1. Establishment of the middle cerebral artery occlusion (MCAO) mouse model

[0137] After anesthetizing C57BL / 6 mice, the common and external carotid arteries were exposed, and a suture was inserted into the common carotid artery and then into the internal carotid artery to occlude the right MCAO. Throughout the procedure, the mice were placed on a heating blanket to maintain normothermia. After 1.5 hours of occlusion, the suture was removed, reperfusion was performed, and closure was performed.

[0138] 2. In vivo distribution imaging

[0139] The enrichment of SHp-EVs with different binding yields in the brains of MCAO mice was investigated. 24 hours after mouse modeling, SHp-EVs obtained by binding EVs with 0.1, 0.5, 1, 5, 10, and 25 μM SHp-PEG-DSP according to Example 3 were labeled with DIR. Each mouse was injected with the same mass of SHp-EVs (200 μL) via the tail vein, and fluorescent images of the mice were captured using an in vivo imaging system.

[0140] The results are as follows Figure 8 ,9 shown, Figure 8 The in vivo distribution images of SHp-EVs injected for 24 hours were obtained by coupling different concentrations of SHp-PEG-DSPE with EVs. Figure 9 for Figure 8 Fluorescence quantitative results of the brains of mice in each group.

[0141] The results showed that SHp-EVs obtained by combining 5 μM SHp-PEG-DSP with EVs had the strongest enrichment effect in the brains of MCAO mice, rather than SHp-EVs prepared by 1.0 μM SHp-PEG-DSP with the highest binding yield.

[0142] At the same time, this example used the above method to study the in vivo distribution of EVs and SHp-EVs (prepared with 5 μM SHp-PEG-DSP, 200 μL) in MCAO mice.

[0143] The results are as follows Figure 10-13 As shown, Figure 10 The brain fluorescence images of EVs and SHp-EVs at 3 and 12 hours. Figure 11 for Figure 10 The quantitative results of brain fluorescence, Figure 12 Fluorescence imaging of EVs and SHp-EVs in brain tissue at 3 and 12 hours. Figure 13 for Figure 12 Fluorescence quantitative value.

[0144] The results showed that SHp-EVs showed faster tropism and higher enrichment in the brain of MCAO mice compared with EVs.

[0145] 3. Therapeutic effects of EVs and SHp-EVs

[0146] (1) TTC staining

[0147] After 24 hours of treatment with EVs and SHp-EVs (5 μM, 200 μL), MCAO mice were sacrificed, their brains removed by rapid decapitation, and the whole brains quickly frozen at -20°C. After 20 minutes, the brains were removed and sectioned coronally, with each slice approximately 2 mm thick. Subsequently, the brain slices were placed in a 2% TTC solution, incubated in a dark water bath at 37°C for 30 minutes, and then fixed in 4% paraformaldehyde for 24 hours. After photographing, the infarct volume was calculated using Image J software. The percentage of infarct volume was calculated as (sum of infarct areas in each layer / sum of areas of each layer) × 100.

[0148] The results are as follows Figure 14-15 As shown, Figure 14 For TTC dyed photos, Figure 15 For infarct area statistics, the results showed that EVs and SHp-EVs significantly reduced the area of ​​cerebral infarction.

[0149] (2) Behavioral science

[0150] Gait footprint test

[0151] The left foot of the treated MCAO mice was smeared with black ink and the right foot was smeared with red ink. The mice were then allowed to start from the same starting point on an A4 white paper and walk freely. The gait behavior of the mice was observed and recorded.

[0152] The open field test was conducted in an open field box measuring 50 cm long, 50 cm wide, and 50 cm high. The box's floor was divided into a central zone and a peripheral zone. Initially, treated MCAO mice were placed in the same position on the peripheral zone of the open field box floor, and their activity was observed over a 10-minute period. Software was used to record the total distance traveled, the distance traveled in the central zone, the percentage of distance traveled in the central zone, and the percentage of time spent in the central zone. After each mouse was tested, the inside of the box was wiped with 75% alcohol to eliminate any residual body odor. The next mouse was tested after the box was dry.

[0153] The results are as follows Figure 16-18 As shown, Figure 16 is the gait footprint diagram, Figure 17 The results of the open field experiment are: Figure 18 for Figure 17 The graph shows the time spent in the center, total distance traveled, and distance traveled in the center area. AD represents the distance traveled in the center area, the total distance traveled, the ratio of the center area to the total distance traveled, and the ratio of time spent in the center area. The results indicate that EVs and SHp-EVs improved the behavior of MCAO mice.

[0154] (3) Biochemical indicators and protein expression

[0155] Enzyme-linked immunosorbent assay (ELISA): Weigh 50 mg of brain tissue and add 300 μL of 4°C PBS. Disperse the tissue using an electric homogenizer, and collect the supernatant. ELISA kits (purchased from Fancovi) were used according to the manufacturer's instructions to measure TNF-α, IL-6, and IL-1β levels.

[0156] Polymerase chain reaction (PCR): 40 mg of brain tissue was weighed from each mouse, added to 500 μL of lysis buffer, and homogenized using an electric homogenizer. The supernatant was collected and RNA was extracted using an RNA extraction kit. RNA was reverse-transcribed into cDNA using a reverse transcription kit and subjected to quantitative PCR. Finally, the gene levels of TNF-α, IL-6, IL-1β, Bax, and Bcl2 were measured using a real-time fluorescence quantitative PCR instrument.

[0157] Western blotting: 80 mg of brain tissue was weighed and 800 μL of protein lysis buffer was added to extract total protein. Protein concentration was determined by the BCA method. The protein sample was added to sample buffer, heat-denatured, and then loaded onto an SDS-PAGE gel for electrophoresis to separate the proteins. The protein was transferred from the gel to a PVDF membrane using a membrane transfer apparatus, and unbound protein sites on the membrane were blocked using a rapid blocking solution. The membrane was then incubated with the primary antibody at 4°C overnight to allow the antibody to bind to the target protein. The membrane was washed to remove unbound primary antibody, and an HRP horseradish peroxidase-labeled secondary antibody was added and incubated at room temperature for 1 hour to assist in the detection of the target protein. The membrane was washed to remove unbound secondary antibody, and then the membrane was placed in a dual-color infrared laser imaging system for protein band imaging.

[0158] The results are as follows Figure 19-22 As shown, Figure 19 The ELISA method was used to measure the levels of inflammatory factors in brain tissue, where A, B, and C were TNF-α, IL-6, and IL-1β, respectively. Figure 20 Representative cytokines were detected by qPCR in RNA extracted from brain tissue, where AL represents the relative mRNA expression levels of TNF-α, IL-6, IL-1β, Bax, Bxl2, JIP1, JIP2, MEKK4, MEKK7, MEKK1, JNK, and P38, respectively. Figure 21 is a representative WB image. Figure 22 for Figure 21 The expression levels of each protein in , where AC are MEEK1, JNK, and P38, respectively.

[0159] The results showed that EVs and SHp-EVs reduced the levels of inflammatory factors TNF-α, IL-6 and IL-1β and apoptotic factor Bax in brain tissue, increased the level of anti-apoptotic factor Bcl-2, and downregulated the expression of genes related to phosphorylated proteins and their corresponding proteins MEKK1, JNK and p38.

[0160] (4) Tissue sections

[0161] Hematoxylin and eosin (H&E) staining: After the animal experiment, mice were anesthetized, decapitated, and brains removed. After washing with saline, the brain tissue was fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned. Paraffin sections were dewaxed, stained with hematoxylin and eosin, dehydrated, and sealed with neutral gum. Under a microscope, cell nuclei appear blue, and cytoplasm appears red.

[0162] Nissl staining: After dewaxing, paraffin sections are stained with 0.5% toluidine blue, dehydrated, and sealed with neutral resin before observation under a microscope. Nissl bodies within neurons appear as dark blue granules, while nuclei appear light blue against a light blue background.

[0163] The results are as follows Figure 23 ,24, Figure 23 For H&E stained sections, Figure 24 Nissl-stained sections. The H&E and Nissl tissue staining results showed that SHp-EVs significantly alleviated neuronal apoptosis.

[0164] Overall, compared with EVs, SHp-EVs showed stronger therapeutic effects, which clarified that they could treat ischemic stroke by regulating MAPK and TNF pathways, thereby exerting anti-inflammatory, anti-oxidative damage and anti-apoptotic effects.

[0165] Although specific embodiments of the present invention have been described above, those skilled in the art will appreciate that these are merely illustrative and that the scope of the present invention is defined by the appended claims. Those skilled in the art may make various changes or modifications to these embodiments without departing from the principles and essence of the present invention, and such changes and modifications are intended to fall within the scope of the present invention.

Claims

1. A targeted extracellular vesicle, characterized in that The invention comprises Yulangsan extracellular vesicles and cerebral ischemia homing peptide, wherein the Yulangsan extracellular vesicles and the cerebral ischemia homing peptide are directly coupled or coupled through a phospholipid-PEG conjugate.

2. The targeted extracellular vesicle according to claim 1, wherein The Yulangsan extracellular vesicles and the cerebral ischemia homing peptide are coupled via Mal-PEG-DSPE. Preferably, the number average molecular weight of the Mal-PEG-DSPE is 1900-2100 Da, for example, 2000 Da.

3. The targeted extracellular vesicle according to claim 1, wherein The Yulangsan extracellular vesicles are prepared by the following method: Take Yulangsan, add PBS solution to homogenize, coarse filter, take the filtrate and ultracentrifuge to remove cell debris, take the filtrate and high-speed centrifuge again to obtain a precipitate, which is the Yulangsan extracellular vesicle.

4. The targeted extracellular vesicle according to claim 3, wherein The preparation method of the Yulangsan extracellular vesicles meets at least one of the following conditions: (1) The Yulang umbrella is an undried fresh Yulang umbrella; (2) The PBS solution is 0.01±0.005M, pH=7.2-7.4; (3) The PBS solution is pre-cooled to 2-6°C before homogenization, preferably to 4°C; (4) The coarse filtration is performed through gauze; (5) The ultracentrifugation and high-speed centrifugation are both performed at 4-25°C, preferably 4°C; (6) The ultracentrifugation is performed according to the following program: 500±200×g for 10±5 min, 3,000±1,000×g for 30±10 min, 10,000±2,000×g for 50±10 min, and 40,000±10,000×g for 40±10 min; preferably, the ultracentrifugation is performed according to the following program: 500×g for 10 min, 3,000×g for 30 min, 10,000×g for 50 min, and 40,000×g for 40 min. (7) The high-speed centrifugation conditions are: 130,000±30,000×g for 80±20 min; preferably 130,000×g for 80 min; (8) After high-speed centrifugation, the precipitate was resuspended in PBS solution and stored at -80°C.

5. A method for preparing targeted extracellular vesicles, characterized in that: The following steps are involved: The Yulangsan extracellular vesicles are taken, and the Yulangsan extracellular vesicles and the cerebral ischemia homing peptide are directly coupled or coupled through a phospholipid-PEG conjugate by using a click chemistry method, a carbodiimide method or a lipid insertion method.

6. The method for preparing targeted extracellular vesicles according to claim 5, wherein: Meet at least one of the following conditions: (1) The Yulangsan exosomes are prepared by the method for preparing the Yulangsan exosomes in the targeted exosomes of claim 3 or 4; (2) The Yulangsan extracellular vesicles and the brain ischemia homing peptide are coupled by click chemistry, and the specific method is as follows: take a Yulangsan extracellular vesicle solution, add dibenzocyclooctylthio-N-hydroxysuccinimide ester solution, mix, react, and modify the surface of the Yulangsan extracellular vesicles with dibenzocyclooctyne groups, and then drop the azide-modified brain ischemia homing peptide to react and couple; preferably, the azide-modified brain ischemia homing peptide is prepared by the following method: dissolve the SHp peptide in PBS and transfer it to a sodium borate buffer solution, add azide-polyethylene glycol-N-hydroxysuccinimide ester, add glycine to terminate the reaction after the reaction, add acetone to the reaction system, centrifuge, and collect the precipitate to obtain the azide-modified brain ischemia homing peptide; (3) The Yulangsan extracellular vesicles and the cerebral ischemia homing peptide are coupled by the carbodiimide method, and the specific method is as follows: taking the Yulangsan extracellular vesicle solution, adding carbodiimide and N-hydroxysuccinimide, mixing, reacting to activate the carboxyl groups on the surface of the Yulangsan extracellular vesicles, and then dripping the cerebral ischemia homing peptide to react and couple; (4) The Yulangsan extracellular vesicles and the cerebral ischemia homing peptide are coupled via Mal-PEG-DSPE, and the specific method is as follows: Preparation of SHp-PEG-DSPE: Mal-PEG-DSPE is dissolved in an organic solvent to obtain solution A, and brain ischemia homing peptide is dissolved in PBS to obtain solution B. Solutions A and B are mixed and reacted to covalently link the brain ischemia homing peptide to the maleimide group of Mal-PEG-DSPE via the thiol group of cysteine ​​to obtain SHp-PEG-DSPE; Preparation of SHp-EVs: Take the Yulangsan extracellular vesicle solution, add the SHp-PEG-DSPE, and carry out coupling reaction to obtain SHp-EVs.

7. The method for preparing targeted extracellular vesicles according to claim 6, wherein: The Yulangsan extracellular vesicles and the cerebral ischemia homing peptide are coupled via Mal-PEG-DSPE and meet at least one of the following conditions: (1) In the step of preparing SHp-PEG-DSPE, the number average molecular weight of the Mal-PEG-DSPE is 1900-2100 Da, for example, 2000 Da; (2) In the step of preparing SHp-PEG-DSPE, the concentration of Mal-PEG-DSPE in the solution A is 3.8-4.1 mg / mL, for example, 4 mg / mL; (3) In the step of preparing SHp-PEG-DSPE, the concentration of the cerebral ischemia homing peptide in the solution B is 10-12 mg / mL, for example, 10 mg / mL; (4) In the step of preparing SHp-PEG-DSPE, the mass ratio of Mal-PEG-DSPE to cerebral ischemia homing peptide is 1:1.5-1:2.8, for example, 1:2.5; (5) In the step of preparing SHp-PEG-DSPE, the organic solvent is N,N-dimethylformamide; (6) In the step of preparing SHp-PEG-DSPE, the PBS is a 0.01±0.005M PBS solution with a pH of 7.4; (7) In the step of preparing SHp-PEG-DSPE, the reaction is carried out under the protection of an inert gas, such as nitrogen; (8) In the step of preparing SHp-PEG-DSPE, the reaction is carried out at 10-40°C, for example, 20-30°C; (9) In the step of preparing SHp-PEG-DSPE, the reaction is carried out under light-proof conditions; (10) In the step of preparing SHp-PEG-DSPE, the reaction time is 4-12 hours, for example, 8 hours; (11) After the step of preparing SHp-PEG-DSPE, a purification step is further included, wherein the purification step comprises dialyzing SHp-PEG-DSPE with water and freeze-drying for storage. Preferably, the dialysis uses a dialysis membrane with a molecular weight cutoff of 3.5 kDa; (12) In the step of preparing SHp-EVs, the concentration of the Yulangsan extracellular vesicles is 0.1-1 mg / mL, for example, 0.5 mg / mL; (13) In the step of preparing SHp-EVs, the reaction concentration of SHp-PEG-DSPE is 0.1-25 μM, preferably 1-5 μM, and more preferably 5 μM; (14) In the step of preparing SHp-EVs, the dosage ratio of the Yulangsan extracellular vesicles to the SHp-PEG-DSPE is 0.5 mg / mL:0.1-25 μM, preferably 0.5 mg / mL:1-5 μM, and more preferably 0.5 mg / mL:5 μM; (15) In the step of preparing SHp-EVs, the temperature of the coupling reaction is 4-10°C, for example, 4°C; (16) In the step of preparing SHp-EVs, the coupling reaction time is 12-24 h; (17) After the step of preparing SHp-EVs, the method further includes a step of removing impurities, wherein the impurity removal step is to remove unreacted raw materials from the coupling reaction product by ultrafiltration; preferably, the ultrafiltration method is to use an ultrafiltration tube with a molecular weight cutoff of 100 kDa, centrifuge at 14,000 × g for 10 minutes, and repeat 3 times.

8. The targeted extracellular vesicles prepared by the preparation method according to any one of claims 5 to 7.

9. Use of the targeted extracellular vesicle according to any one of claims 1 to 4 and 8 in the preparation of a medicament for improving cerebral ischemia.

10. The use according to claim 9, characterized in that The drug for improving cerebral ischemia is used to treat ischemic stroke; Preferably, the targeted extracellular vesicles improve cerebral ischemia symptoms by reducing inflammatory factors, apoptotic factors and / or phosphorylated proteins in brain tissue and increasing anti-apoptotic factors; More preferably, the inflammatory factors are TNF-α, IL-6 and / or IL-1β; the apoptotic factor is Bax, the anti-apoptotic factor is Bcl-2, and the phosphorylated proteins are MEKK1, JNK and P38.

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