Use of an engineered extracellular vesicle in the preparation of a product for preventing or treating nerve injury

Abstract

The application provides application of an engineered extracellular vesicle in preparation of a product for preventing and treating nerve injury, and belongs to the technical field of prevention and treatment of nerve injury. The application provides the application of the engineered extracellular vesicle in preparation of the product for preventing and treating nerve injury, wherein the engineered extracellular vesicle is an extracellular vesicle loaded with brain-derived neurotrophic factor and brain-targeting peptide, the extracellular vesicle is derived from human nasal mucosa mesenchymal stem cells, and the brain-targeting peptide is RVG 29 peptide, and the amino acid sequence is shown in SEQ ID NO. 1. The application can significantly improve brain targeting and drug utilization rate, and the nerve repair effect is multi-dimensional and remarkable.

Description

Technical Field

[0001] This invention belongs to the field of nerve injury prevention and treatment technology, specifically relating to the application of engineered extracellular vesicles in the preparation of products for the prevention and treatment of nerve injury. Background Technology

[0002] Manganese is an essential trace element for the human body, participating in various physiological processes such as enzyme catalysis, neurotransmitter metabolism, and bone development. However, long-term or high-concentration exposure to manganese, especially occupational exposure or environmental pollution, can easily lead to abnormal accumulation of manganese in the brain, inducing neurological damage, manifesting as Parkinson's Disease (PD)-like motor disorders, cognitive decline, and other symptoms, resulting in manganese poisoning. The core mechanisms of manganese-induced neurological damage include dopaminergic neuron damage, neurogenesis disorders, and neuroinflammatory responses; these pathological changes are often difficult to reverse. Currently, clinical treatment mainly uses metal chelating agents such as calcium edetate sodium (CaNa2-EDTA), whose mechanism of action is mainly to promote manganese excretion. However, this approach has significant limitations: chelating agents can only reduce manganese load and cannot directly repair nerve damage, leading to persistent functional impairment in patients. Furthermore, existing neurorepair strategies lack targeted and multidimensional intervention capabilities, failing to systematically address the problem of manganese-induced neurological damage.

[0003] Mesenchymal stem cells (MSCs) are considered an ideal cell type for research on neurological diseases due to their self-renewal, multi-lineage differentiation capabilities, and low immunogenicity. Human nasal mucosal mesenchymal stem cells (hnmMSCs), as a novel type of MSC, exhibit high proliferation efficiency, strong self-renewal capacity, and short passage time, and readily differentiate into dopaminergic neurons, making them an ideal cell source for treating neurodegenerative diseases. The therapeutic effect of hnmMSCs primarily derives from the extracellular vesicles (EVs) released by their paracrine signaling. EVs possess the ability to carry bioactive substances such as proteins and nucleic acids, participate in intercellular communication, and maintain the functional characteristics of the source cells. However, conventional hnmMSC-derived EVs have significant limitations in treatment. During in vivo distribution, EVs tend to accumulate in non-target organs such as the liver and spleen, exhibiting limited targeting ability in the brain, thus restricting their therapeutic efficiency in central nervous system diseases. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide the application of engineered extracellular vesicles in the preparation of products for the prevention and treatment of nerve damage, which can significantly improve brain targeting and drug utilization, and has a multidimensional and significant effect on nerve repair.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the application of engineered extracellular vesicles (abbreviated as: BDNF@RVG-EVs) in the preparation of products for preventing and treating nerve damage. The engineered extracellular vesicles are extracellular vesicles loaded with brain-derived neurotrophic factor (BDNF) and a brain-targeting peptide. The extracellular vesicles are derived from human nasal mucosal mesenchymal stem cells, and the brain-targeting peptide is RVG. 29 The peptide has the amino acid sequence shown in SEQ ID NO.1.

[0006] This invention also provides an application of engineered extracellular vesicles (abbreviated as: BDNF@RVG-EVs) in the preparation of any of the following products: (1) promoting neurogenesis; (2) repairing dopaminergic neurons; (3) reducing neuroinflammation; wherein the engineered extracellular vesicles are extracellular vesicles loaded with BDNF and brain-targeting peptides, wherein the extracellular vesicles are derived from human nasal mucosal mesenchymal stem cells, and the brain-targeting peptides are RVG 29 The peptide has the amino acid sequence shown in SEQ ID NO.1.

[0007] This invention also provides the application of engineered extracellular vesicles (abbreviated as: BDNF@RVG-EVs) in the preparation of products for treating Parkinson's-like nerve injuries. The engineered extracellular vesicles are extracellular vesicles loaded with brain-derived neurotrophic factor and brain-targeting peptides. The extracellular vesicles are derived from human nasal mucosal mesenchymal stem cells, and the brain-targeting peptide is RVG. 29 The peptide has the amino acid sequence shown in SEQ ID NO.1.

[0008] Preferably, the nerve damage includes nerve damage caused by manganese.

[0009] Preferably, the Parkinsonian nerve injury includes Parkinsonian nerve injury caused by manganese exposure.

[0010] Preferably, the formulation type of the product is a nasal formulation.

[0011] Preferably, the nasal preparation includes nasal drops.

[0012] Preferably, the method for preparing the engineered extracellular vesicles includes the following steps: DSPE-PEG... 2000 -RVG 29 DSPC and cholesterol were dissolved in a methanol-chloroform mixture and rotary evaporated until a film was formed. The film was mixed with a PBS solution containing brain-derived neurotrophic factor (BDNF), sonicated in a water bath, extruded using a filter membrane, and ultrafiltered to obtain a film loaded with BDNF and DSPE-PEG. 2000 -RVG 29Liposomes were mixed with extracellular vesicles derived from human nasal mucosal mesenchymal stem cells, and after sonication in an ice bath, the mixture was extruded using a filter membrane and ultrafiltered to obtain engineered extracellular vesicles.

[0013] Preferably, the method for preparing extracellular vesicles derived from human nasal mucosal mesenchymal stem cells includes the following steps: after adhering to the primary culture of human nasal polyp tissue, human nasal mucosal mesenchymal stem cells are obtained; after 3D dynamic culture of human nasal mucosal mesenchymal stem cells, the supernatant is taken and the extracellular vesicles derived from human nasal mucosal mesenchymal stem cells are separated by differential centrifugation; the differential centrifugation method includes the following steps: the supernatant is centrifuged sequentially at 200~800g for 3~20min, at 1000~5000g for 10~60min, at 8000~150000g for 30min, and at 100000~200000g for 70~120min.

[0014] The beneficial effects of this invention are: This invention provides, for the first time, the application of engineered extracellular vesicles in the preparation of products for the prevention and treatment of nerve damage, wherein engineered extracellular vesicles can significantly improve brain targeting and drug utilization: This invention utilizes RVG 29 The combination of peptide modification and nasal instillation delivery technology significantly enhances the ability of extracellular vesicles to cross the blood-brain barrier. RVG 29 Peptides can specifically recognize acetylcholine receptors on neuronal membranes, enabling precise targeting of brain tissue. Nasal instillation effectively delivers the drug to the central nervous system, avoiding gastrointestinal degradation and the first-pass effect in the liver, thus significantly increasing the effective concentration and bioavailability of the drug in the brain. This combined strategy allows extracellular vesicles to accumulate significantly in key brain regions, providing an efficient central delivery route for nerve injury repair.

[0015] Furthermore, the application described in this invention has the advantage of multidimensional and significant neurorepair effects: the BDNF@RVG-EVs system of this invention exhibits multi-level neuroprotective and repair effects in manganese-induced nerve injury. Unlike existing chelating agents (such as CaNa2-EDTA), which can only promote the excretion of metal ions and are difficult to directly repair nerve damage, the engineered extracellular vesicles of this invention can deliver BDNF, simultaneously promoting the proliferation and differentiation of neural stem cells, repairing the function of dopaminergic neurons, inhibiting neuroinflammatory responses, and improving motor disorders caused by manganese exposure. The engineered extracellular vesicles provided by this invention achieve a synergistic effect of structural remodeling, functional recovery, and inflammatory regulation, demonstrating a comprehensive neurorepair advantage significantly superior to existing treatment methods. Attached Figure Description

[0016] Figure 1 The TEM results are for the engineered extracellular vesicles obtained in Example 1; Figure 2 The left and right figures show the hydration particle size distribution and zeta potential results of the engineered extracellular vesicles obtained in Example 1. Figure 3 The fluorescence distribution in different groups after intranasal administration; Figure 4 The results show the fluorescence distribution of different brain regions after paraffin sections of brain tissue. A represents the olfactory bulb region, B represents the lateral ventricle and striatum region, and C represents the hippocampus region. Figure 5 The results are for the rotating bar experiment and the open field experiment. A represents the results of the rotating bar experiment, B represents the statistical results of the average velocity and rest time in the open field experiment, and C represents the results of the motion trajectory in the open field experiment. * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001. Figure 6 The results show the repair effect of BDNF@RVG-EVs on dopaminergic neurons in manganese-exposed mice; Figure 7 Immunofluorescence results of the effects of BDNF@RVG-EVs on neurogenesis in manganese-exposed mice: A represents the results of SOX2, a neural stem cell marker in the lateral ventricle; B represents the results of DCX, a marker of immature neurons in the lateral ventricle; C represents the results of SOX2, a neural stem cell marker in the hippocampus; and D represents the results of DCX, a marker of immature neurons in the hippocampus. Figure 8 The results of RT-qPCR on the effects of BDNF@RVG-EVs on neurogenesis in manganese-exposed mice are shown. The first row shows the results for the lateral ventricle, and the second row shows the results for the hippocampus. * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001. Figure 9 Immunofluorescence results of BDNF@RVG-EVs on neuroinflammation in manganese-exposed mice, where A represents the results in the lateral ventricle and B represents the results in the hippocampus; Figure 10 The results of RT-qPCR detection of the effect of BDNF@RVG-EVs on neuroinflammation in manganese-exposed mice are shown. The first row shows the results for the lateral ventricle, and the second row shows the results for the hippocampus. * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001. Detailed Implementation

[0017] This invention provides an application of engineered extracellular vesicles (abbreviated as: BDNF@RVG-EVs) in any of the following: (1) preparing products for preventing and treating nerve damage; (2) preparing products for promoting neurogenesis; (3) preparing products for repairing dopaminergic neurons; (4) preparing products for treating neuroinflammation; (5) preparing products for treating Parkinson's-like nerve damage; wherein the engineered extracellular vesicles are loaded with BDNF and connected to RVG 29 Extracellular vesicles of peptides, said extracellular vesicles derived from human nasal mucosal mesenchymal stem cells, said RVG 29 The amino acid sequence of the peptide is YTIWMPENPRPGTPCDIFTNSRGKRASNG, as shown in SEQ ID NO.1.

[0018] In this invention, BDNF plays a crucial role in the development of the nervous system, promoting neurogenesis and the repair of dopaminergic neurons. BDNF@RVG-EVs are based on extracellular vesicles (EVs) derived from human nasal mucosal mesenchymal stem cells (hnmMSCs), modified with rabies virus glycoprotein (RVG) peptides mediated by DSPE-PEG, enabling EVs to possess highly efficient brain-targeting capabilities, especially enabling them to enter the brain through the olfactory and trigeminal nerve pathways and accumulate in key areas of nerve damage.

[0019] This invention utilizes EVs derived from hnmMSCs, which, compared to existing cell-engineered exosomes in the field, possess more natural neural repair properties and the advantage of stem cell origin. In terms of delivery strategy, this invention employs DSPE-PEG-mediated RVG... 29 Peptide functionalization, combined with the olfactory and trigeminal pathways, enables highly efficient brain-targeted delivery of exosomes (EVs), which accumulate in key areas of nerve injury, significantly improving intrabrain delivery efficiency. Functionally and in terms of mechanism of action, the EVs of this invention not only deliver BDNF but also possess the inherent neurorepair potential of hnmMSCs, enabling multi-dimensional intervention: promoting dopaminergic neuron repair, activating neurogenesis, and inhibiting neuroinflammation, while balancing targeted and functional repair capabilities. Furthermore, this invention, through material selection, targeted delivery strategies, and functional repair integration, forms a comprehensive technical solution of "brain-targeted delivery + multi-dimensional neurorepair," distinguishing it from existing single-action modes that rely solely on genetically engineered exosomes.

[0020] This invention uses specific embodiments to verify its effectiveness. Results show that after intervention with BDNF@RVG-EVs in mice with manganese-induced neurological injury, the brain distribution of BDNF@RVG-EVs was significantly enhanced, especially in key areas of neurological injury such as the hippocampus and striatum, thus significantly improving treatment efficiency. In the application described in this invention, BDNF@RVG-EVs can improve Parkinson's-like neurological injury in manganese-exposed mice. This superior effect relies on the comprehensive verification of multiple techniques: immunofluorescence and RT-qPCR detection of neurogenic markers, dopaminergic neuronal repair-related proteins, and inflammatory factor expression, combined with behavioral experiments (such as the rotarod test and open field test) to comprehensively assess motor coordination and symptom improvement, demonstrating the significant effect of BDNF@RVG-EVs on neurological repair. Based on a thorough analysis of the shortcomings of existing technologies and the pathological mechanism of manganese-induced neurological injury, this invention proposes a novel treatment strategy combining brain targeting and functional repair. This strategy not only solves the problem of low brain delivery efficiency of conventional EVs but also innovatively achieves multidimensional comprehensive intervention for manganese-exposed neurological injury, providing a new technical approach for the treatment of related diseases.

[0021] While other methods exist for central drug delivery or neurotrophic agents, such as conventional liposomes, polymer nanoparticles, or nasal spray carriers, these alternatives have significant limitations in terms of blood-brain barrier crossing efficiency, targeting, and biosafety. Traditional liposomes and polymer nanocarriers are easily cleared by the reticuloendothelial system, making precise delivery to the brain difficult; while ordinary extracellular vesicles, although biocompatible, lack specific recognition capabilities, resulting in limited drug accumulation in brain tissue. Furthermore, BDNF protein administered nasally alone is prone to rapid degradation and inefficient targeting of brain tissue. This invention utilizes RVG... 29 Peptide-modified extracellular vesicles combined with nasal instillation offer a combination of high targeting, stability, and delivery efficiency, making it the most effective and innovative approach for brain nerve repair. Existing technologies struggle to achieve the combined effects of this invention.

[0022] In this invention, the nerve damage preferably includes nerve damage caused by manganese; the Parkinsonian nerve damage preferably includes Parkinsonian nerve damage caused by manganese exposure; the formulation type of the product is preferably a nasal formulation, and the nasal formulation preferably includes nasal drops.

[0023] In this invention, the method for preparing the engineered extracellular vesicles includes the following steps: DSPE-PEG... 2000 -RVG 29DSPC and cholesterol were dissolved in a methanol-chloroform mixture and rotary evaporated until a film was formed. The film was mixed with a PBS solution containing brain-derived neurotrophic factor (BDNF), sonicated in a water bath, extruded using a filter membrane, and ultrafiltered to obtain a film loaded with BDNF and DSPE-PEG. 2000 -RVG 29 Liposomes were mixed with extracellular vesicles derived from human nasal mucosal mesenchymal stem cells, and after sonication in an ice bath, the mixture was extruded using a filter membrane and ultrafiltered to obtain engineered extracellular vesicles.

[0024] This invention relates to DSPE-PEG 2000 -RVG 29 The specific sources of DSPC and cholesterol are not particularly limited; commercially available products commonly used in the field are acceptable. In this invention, DSPE-PEG... 2000 -RVG 29 The preferred mass ratio of DSPE to DSPC is 3:1 to 1:5, more preferably 3:4; DSPE-PEG 2000 -RVG 29 The mass ratio of liposomes to cholesterol is preferably 5:1 to 1:5, more preferably 3:1; the volume ratio of methanol to chloroform in the methanol-chloroform mixture is preferably 5:1 to 1:5, more preferably 1:1; the pore size of the filter membrane is preferably 50 to 500 nm, more preferably 200 nm; and the extrusion cycle is preferably 10 to 30 times, more preferably 20 times. In this invention, the rotary evaporation temperature is preferably 37°C, the rotary evaporation speed is preferably 110 rpm, and the rotary evaporation time is preferably 1 hour. In this invention, the mass ratio of liposomes to extracellular vesicles derived from human nasal mucosal mesenchymal stem cells is preferably 4:1. In this invention, the water bath ultrasound temperature is preferably 20~28℃, more preferably 25℃; the water bath ultrasound power is preferably 50~500W, more preferably 100W; the water bath ultrasound time is preferably 1~30min, more preferably 10min; the ice bath ultrasound power is preferably 50~500W, more preferably 100W; the ice bath ultrasound time is preferably 1~30min, more preferably 5min; the ultrafiltration is performed using a 100KDa ultrafiltration tube, and the centrifugation parameters are preferably 3000~8000rpm for 5~30min, more preferably 4000~7000rpm for 10~20min.

[0025] In this invention, the preferred method for preparing extracellular vesicles derived from human nasal mucosal mesenchymal stem cells includes the following steps: after adhering to a wall for primary culture of human nasal polyp tissue, human nasal mucosal mesenchymal stem cells are obtained; after 3D dynamic culture of human nasal mucosal mesenchymal stem cells, the supernatant is taken and the extracellular vesicles derived from human nasal mucosal mesenchymal stem cells are separated by differential centrifugation; the differential centrifugation method includes the following steps: the supernatant is centrifuged sequentially at 200~800g for 3~20min, at 1000~5000g for 10~60min, at 8000~150000g for 30min, and at 100000~200000g for 70~120min.

[0026] This invention does not specifically limit the source of human nasal polyp tissue. After obtaining human nasal polyp tissue, it is preferable to wash it first. The washing reagent is preferably a PBS solution containing penicillin-streptomycin and amphotericin B, wherein the volume concentration of penicillin-streptomycin is preferably 1%, and the final concentration of amphotericin B is preferably 1%. After washing, the tissue is cut into pieces, preferably 1 mm pieces, and then subjected to adherent primary culture. The culture medium used for adherent primary culture is preferably composed of DMEM-F12, FBS, penicillin-streptomycin, and amphotericin B, wherein the volume percentages of DMEM-F12, FBS, penicillin-streptomycin, and amphotericin B are preferably 88%, 10%, 1%, and 1%, respectively. The culture medium is changed every 1-3 days. When the fibroblast-like cells reach 70-80%, human nasal mucosal mesenchymal stem cells are obtained. After successfully obtaining hnmMSCs, 3D culture was performed, preferably using the 3D FloTrix® miniSPIN bioreactor system for dynamic 3D culture. The stirring speed was preferably 35~50 rpm / min, more preferably 40~45 rpm / min, until the cell culture density reached 5×10⁶ cells / min. 5 When separating individual cells / microcarriers, the supernatant is collected and separated using differential centrifugation. The preferred differential centrifugation method includes the following steps: centrifuging the supernatant sequentially at 200-800g for 3-20 min, 1000-5000g for 10-60 min, 8000-150000g for 30 min, and 100000-200000g for 70-120 min; more preferably, centrifuging sequentially at 300g for 10 min, 2000g for 20 min, 10000g for 30 min, and 120000g for 70 min. The differential centrifugation is preferably performed entirely at 4°C.

[0027] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0028] Unless otherwise specified, the following embodiments are all conventional methods.

[0029] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0030] Example 1 Preparation of engineered extracellular vesicles BDNF@RVG-EVs (1) Preparation of EVs derived from hnmMSCs: Human nasal polyp tissue was washed with PBS containing penicillin-streptomycin and amphotericin B (volume concentration of 1%). The tissue was then cut into 1 mm pieces and cultured in 6-well plates. The culture medium consisted of DMEM-F12, FBS, penicillin-streptomycin, and amphotericin B, with a volume percentage of 88%:10%:1%:1%. The medium was changed daily. After 21 days of culture, fibroblast-like cells reached 70%–80%, yielding hnmMSCs.

[0031] hnmMSCs were induced to undergo osteogenic and adipogenic differentiation by culturing them in osteogenic and adipogenic differentiation media for 3 weeks. After 3 weeks, the results of adipogenic and osteogenic differentiation were identified by staining with a low-glucose DMEM-LG basal medium, with dexamethasone, ascorbic acid, and sodium glycerophosphate added to final concentrations of 10 nM, 50 μM, and 10 mM, respectively. Alizarin Red staining: When the cells reached a certain degree of confluence, the old medium was discarded, and the cells were cultured in fresh osteogenic differentiation medium for 3 weeks. The original differentiation medium was then aspirated, and the cells were washed several times with PBS. Cells were fixed with 4% neutral formaldehyde solution. After half an hour, the neutral formaldehyde solution was aspirated, and the cells were washed again with PBS. 1 mL of alizarin red dye solution was added to each well of a six-well plate. After 3-5 minutes, the staining solution was aspirated, and the cells were washed several times with PBS. The cells were then observed and photographed under a microscope. Oil Red O staining: When cells reach a certain degree of confluence, discard the old culture medium and culture in fresh adipogenic differentiation medium for 3 weeks. Then, aspirate the old differentiation medium and wash repeatedly with PBS. Fix cells with 4% neutral formaldehyde solution. After half an hour, discard the neutral formaldehyde solution and wash again with PBS. Add Oil Red O working solution. After staining for 1 hour, discard the staining solution, wash several times with PBS, and observe and photograph under a microscope.

[0032] Alizarin Red S positive (successful osteogenic differentiation), with red mineralized nodule deposition; Oil Red O positive (successful adipogenic differentiation), with obvious red lipid droplets observed; and when the expression of stem cell markers SSEA-4 and Nanog was stable using immunofluorescence, it indicated that the above steps yielded functionally stable hnmMSCs.

[0033] After successfully obtaining hnmMSCs, 3D dynamic cell culture was performed using the 3D FloTrix® miniSPIN bioreactor system at a cell culture density of 5 × 10⁻⁶. 5 The culture medium used for 3D dynamic culture of individual cells / microcarriers was DMEM-F12 basal medium, with a stirring speed of 35 rpm. The supernatant was collected and separated into EVs derived from hnmMSCs by differential centrifugation. The differential centrifugation method consisted of sequentially centrifuging the supernatant at 200-800g for 3-20 min, 1000-5000g for 10-60 min, 8000-150000g for 30 min, and 100000-200000g for 70-120 min; more preferably, sequentially centrifuging at 300g for 10 min, 2000g for 20 min, 10000g for 30 min, and 120000g for 70 min. The differential centrifugation was preferably performed entirely at 4°C.

[0034] (2) BDNF@RVG-EVs (or abbreviated as B-EVs) RVG Synthesis of ) DSPE-PEG 2000 -RVG 29 (Purchased from Xi'an Ruixi Biotechnology Co., Ltd.), DSPC and cholesterol dissolved in a methanol-chloroform mixture, DSPE-PEG 2000-RVG 29 The amounts of DSPC and cholesterol were 3.6 mg, 4.8 mg, and 1.2 mg, respectively. The volume ratio of methanol to chloroform in the methanol-chloroform mixture was 1:1. The above solution was then rotary evaporated at 37 °C and 110 rpm for 1 h until a film was formed.

[0035] Add 4 mL of PBS solution containing BDNF protein to a round-bottom flask. Mix the membrane with the PBS solution containing brain-derived neurotrophic factor (BDNF). Sonicate the membrane in a 25°C, 100W water bath for 10 min to fully disperse it in the PBS solution. Extrude the membrane using a 200 nm filter membrane for 20 cycles and ultrafilter using a 100 kDa ultrafiltration tube. Centrifuge at 5000 rpm for 10 min, then resuspend in PBS solution to obtain BDNF and DSPE-PEG 2000-RVG. 29 liposomes.

[0036] Liposomes were mixed with EVs derived from hnmMSCs at a mass ratio of 4:1. The mixture was then sonicated in an ice bath at 100 W for 1 second at 1 second intervals for 5 minutes. The mixture was then extruded through a 200 nm filter membrane for 20 cycles, followed by ultrafiltration using a 100 kDa ultrafiltration tube. The mixture was then centrifuged at 5000 rpm for 10 minutes to obtain engineered extracellular vesicle B-EVs. RVG (Or abbreviated as BDNF@RVG-EVs, or abbreviated as LNPs+BDNF+Exo).

[0037] The morphology of the engineered extracellular vesicles was observed using transmission electron microscopy (TEM), and the results are as follows: Figure 1 As shown, the hydration size distribution and zeta potential of engineered extracellular vesicles were then measured, and the results are as follows. Figure 2 As shown.

[0038] Example 2 Distribution of BDNF@RVG-EVs at various sites in mice after intervention with manganese-induced neurological injury The BDNF@RVG-EVs prepared in Example 1 and unmodified EVs (obtained from step (1) of Example 1) were used as experimental materials. The two experimental materials were labeled with DiI fluorescent dye: EVs or BDNF@RVG-EVs were incubated with DiI at 37°C in the dark for 30 min according to the reagent instructions, and the free dye was removed by ultrafiltration to obtain labeled BDNF@RVG-EVs and EVs.

[0039] Establishment of a mouse model of nerve damage induced by chronic manganese exposure: Eight-week-old male C57BL / 6J mice (purchased from a qualified laboratory animal supplier) were selected and began experiments after one week of acclimatization. Manganese chloride (MnCl2, Sigma-Aldrich) solution was administered intranasally at a dose of 20 mg / kg per mouse. The MnCl2 solution was prepared by diluting it to the required concentration with sterile saline. During administration, the mouse's head was tilted at 45°, and the drug was slowly instilled into the nasal cavity using a micropipette, 5 μL per nostril, with approximately 30-second intervals to ensure adequate drug absorption. The mice were given the drug five times a week for four consecutive months to establish a stable chronic manganese-induced neurological injury model. The control group received an equal volume of sterile saline during the same period, and all feeding conditions were kept consistent. The results showed that the chronic manganese exposure mouse model exhibited stable Parkinson's disease-like symptoms and is suitable for subsequent intervention studies.

[0040] After establishing a mouse model of chronic manganese exposure-induced nerve injury, mice were randomly assigned to three groups and administered sterile PBS (control group), DiI-labeled EVs (100 μg / mouse), or DiI-labeled BDNF@RVG-EVs (100 μg / mouse), respectively. Administration was via nasal instillation: 5 μL was instilled into each nostril while the mouse's head was held at a 45° angle, with 30-second intervals until complete absorption. Animal samples were collected at 6 and 12 hours post-administration, and fluorescence distribution in the whole body and major organs (brain, liver, kidney, spleen, lung, and heart) was detected using an IVIS Spectrum imaging system.

[0041] The results are as follows Figure 3 As shown, 6 hours after intranasal administration, DiI-labeled BDNF@RVG-EVs exhibited strong fluorescence signals in the mouse brain, with a higher distribution intensity than EVs at the same dose. At 12 hours, the fluorescence signal of BDNF@RVG-EVs in brain tissue remained at a high level, demonstrating stronger brain tissue enrichment capacity compared to unmodified EVs. Furthermore, the fluorescence signal of BDNF@RVG-EVs in the liver was lower, indicating a relatively limited peripheral distribution.

[0042] Six hours after drug administration, brain tissue was separately harvested for paraffin sectioning, and the fluorescence distribution in different brain regions was analyzed by immunofluorescence staining. The results are as follows: Figure 4 As shown, more DiI markers BDNF@RVG-EVs can be detected in the olfactory bulb and hippocampus regions.

[0043] In summary, after intranasal administration, BDNF@RVG-EVs were able to enter mouse brain tissue and accumulate in specific regions, with a higher distribution level in brain tissue than unmodified EVs.

[0044] Example 3 The effect of BDNF@RVG-EVs on improving motor behavior in mice with manganese-induced neurological injury To evaluate the effects of BDNF@RVG-EVs on motor function in manganese-exposed mice, model mice were selected and subjected to behavioral tests after four interventions. The experiments used included the rotarod test and the open field test. The specific methods are as follows: The method for establishing a mouse model of nerve damage caused by chronic manganese exposure is the same as in Example 2.

[0045] The successfully established manganese-exposed mice were randomly divided into four groups (n=6 / group), and normal control mice that had not been exposed to manganese were set up as the control group. The specific grouping and intervention are as follows: Control group: mice not exposed to manganese were given an equal volume of sterile PBS by nasal drip once a week for 4 consecutive weeks; Mn group: mice exposed to manganese were given an equal volume of sterile PBS by nasal drip once a week for 4 consecutive weeks; BDNF@RVG-EVs intervention group: mice exposed to manganese were given BDNF@RVG-EVs by nasal drip at a dose of 100 μg / mouse (based on the mass of EVs) once a week for 4 consecutive weeks; ordinary EVs intervention group: mice exposed to manganese were given hnmMSC-EVs (prepared by step (1) of Example 1) by nasal drip at a dose of 100 μg / mouse once a week for 4 consecutive weeks; CaNa2-EDTA intervention group: mice exposed to manganese were given CaNa2-EDTA by intraperitoneal injection at a dose of 25 mg / kg / day for 4 consecutive days per week, with an interval of 3 days, for 4 consecutive weeks. The nasal drip method was the same as described in the model construction section. For intraperitoneal injection, the mouse abdomen was disinfected, and the needle was inserted 1 cm to the left of the midline of the abdomen to avoid damage to internal organs. After the intervention, the motor coordination ability and spontaneous activity level of the manganese-exposed mice were tested using the rotarod test and open field test. Immunofluorescence and RT-qPCR technology were used to detect the level of dopamine neurons, neurogenesis, and neuroinflammatory status in the brain. The effects of each intervention were comprehensively evaluated at the molecular, cellular, and behavioral levels to clarify the therapeutic advantages of BDNF@RVG-EVs.

[0046] (1) A rotundus test was performed after each intervention to assess motor coordination and endurance. The experimental equipment was a motor-driven rotating rod with adjustable speed. Mice underwent two days of acclimatization training before the experiment. In the formal experiment, mice were placed on the rotundus with an initial speed of 0 rpm and an acceleration of 5 rpm, and the time it took for the animals to fall off the rotundus was recorded. The equipment was cleaned with 75% alcohol after each experiment.

[0047] The results are as follows Figure 5 As shown in Figure A, in the rotarod experiment, the fall time of manganese-exposed mice was reduced compared to the control group. After three interventions, the fall time of mice in the BDNF@RVG-EVs treatment group was prolonged; after four interventions, the fall time of mice in both the EVs group and the BDNF@RVG-EVs group was prolonged, especially in the BDNF@RVG-EVs group, which was significantly longer than that in the CaNa2-EDTA group.

[0048] (2) An open field test was conducted after four interventions to assess spontaneous activity. The experimental site was a transparent plastic open box with the bottom divided into several compartments. Mice were placed in the center and allowed to move freely, while a camera system recorded their movement trajectory, speed, and dwell time. The box was cleaned with 75% alcohol after each experiment.

[0049] The results are as follows Figure 5As shown in B and C, in the open field experiment, after four interventions, the movement speed of mice in both the EVs group and the BDNF@RVG-EVs group increased, and the resting time decreased. Movement trajectory analysis showed that the activity range of mice in the manganese exposure group was limited to the edge of the open field, while the activity range of mice in the intervention group expanded, and the time spent in the central region increased.

[0050] The combined results indicate that BDNF@RVG-EVs treatment can improve the motor function and exploratory behavior of manganese-exposed mice, and its effect is better than that of unmodified EVs and CaNa2-EDTA.

[0051] (3) BDNF@RVG-EVs promote the repair of dopaminergic neuron damage in manganese-exposed mice Experimental methods: To detect the repair effect of BDNF@RVG-EVs on dopaminergic neurons in manganese-exposed mice, immunofluorescence was used for analysis.

[0052] Immunofluorescence: Mouse brain tissue was collected and fixed in 4% paraformaldehyde to prepare paraffin sections. Sections were hydrated after treatment with xylene and different concentrations of alcohol, followed by antigen retrieval via heating in EDTA buffer (pH=8.0). After washing with PBS, sections were incubated with 3% hydrogen peroxide, then blocked with 10% goat serum. Primary antibody was added and incubated overnight in a humidified chamber at 4°C. After washing with PBS, the appropriate secondary antibody was added and incubated in the dark. Tyramine fluorescein was then added for labeling and DAPI staining. After washing with PBS, the sections were mounted. Finally, images were acquired using a fully automated slide scanning system.

[0053] The results are as follows Figure 6 As shown, the density of tyrosine hydroxylase (TH)-positive fibers decreased in the striatal region of manganese-exposed mice, indicating damage to dopaminergic neurons. After treatment intervention, the density of TH-positive fibers increased compared to the manganese-exposed group, with the BDNF@RVG-EVs group showing more significant recovery. Overall, the results indicate that BDNF@RVG-EVs treatment can improve the damage to striatal DA neurons in manganese-exposed mice, and its effect is superior to that of unmodified EVs.

[0054] (4) BDNF@RVG-EVs promote neurogenesis in manganese-exposed mice Experimental methods: To detect the effect of BDNF@RVG-EVs on neurogenesis in manganese-exposed mice, two methods were used: immunofluorescence and real-time quantitative PCR (RT-qPCR). The immunofluorescence experimental steps were the same as those described in (3) above. Immunofluorescence was used to detect the neural stem cell marker SOX2 and the immature neuron marker DCX, while RT-qPCR was used to detect the expression levels of the Sox2, Nestin, and Dcx genes.

[0055] RT-qPCR: Total RNA was extracted from frozen brain tissue using TRIZOL, and high-purity RNA was obtained by isopropanol precipitation and washing with 75% ethanol. The concentration and purity were then measured using a microplate reader. Subsequently, the RNA was treated with DNase I to remove genomic DNA, and the RNA was reverse transcribed into cDNA using a reverse transcription kit. Finally, using the cDNA as a template, real-time quantitative PCR was performed using the SYBR Green assay. Cyclic and melting curve analysis were performed according to standard procedures, and the results were analyzed using 2... -ΔΔCt The method calculates the relative expression level of the target gene.

[0056] Results: In the lateral ventricles, SOX2-positive cells were distributed along the lateral walls. In all treatment groups, the number of SOX2-positive cells increased compared to the manganese exposure group, with a greater increase in the BDNF@RVG-EVs group. DCX-positive cells also showed an increase, more pronounced in the BDNF@RVG-EVs group, and a tendency for cell migration towards the striatum was observed. In the hippocampus, both SOX2 and DCX-positive cells showed an increasing trend, with a higher number in the BDNF@RVG-EVs group, followed by the EVs group, while the CaNa2-EDTA group showed less change (see...). Figure 7 ).

[0057] RT-qPCR results showed that in the lateral ventricle, Sox2 expression was higher than in the manganese group: an increase of 43.17% in the EVs group, 47.95% in the BDNF@RVG-EVs group, and 34.26% in the CaNa2-EDTA group. Nestin expression also showed an increasing trend: an increase of 47.58% in the EVs group, 48.00% in the BDNF@RVG-EVs group, and 29.33% in the CaNa2-EDTA group. Dcx expression increased in all treatment groups, with an increase of 29.70% in the BDNF@RVG-EVs group, 24.45% in the EVs group, and 3.27% in the CaNa2-EDTA group. In the hippocampus, Sox2 expression was increased: an increase of 22.65% in the EVs group, 35.98% in the BDNF@RVG-EVs group, and 7.88% in the CaNa2-EDTA group. Nestin expression increased: 27.19% in the EVs group, 42.57% in the BDNF@RVG-EVs group, and 16.38% in the CaNa2-EDTA group. Dcx expression increased: 26.08% in the EVs group, 36.03% in the BDNF@RVG-EVs group, and 23.27% in the CaNa2-EDTA group (see...). Figure 8 ).

[0058] The above results indicate that in manganese-exposed mice, the BDNF@RVG-EVs treatment group showed higher levels of neural stem cell and immature neuron marker expression in both the SVZ and hippocampus, and had a stronger neurogenesis-promoting effect compared to the EVs group and the CaNa2-EDTA group.

[0059] (5) BDNF@RVG-EVs inhibited neuroinflammation in manganese-exposed mice Experimental methods: To investigate the effect of BDNF@RVG-EVs on neuroinflammation in manganese-exposed mice, immunofluorescence and RT-qPCR were used to detect the expression levels of inflammation-related factors in the lateral ventricle and hippocampus. Immunofluorescence was used to observe the distribution of TNF-α-positive cells, and RT-qPCR was used to detect the transcriptional levels of the pro-inflammatory factor TNF-α and the anti-inflammatory factors Cd206 and Arg1. The experimental steps were consistent with steps (3) and (4), respectively.

[0060] Experimental results: Immunofluorescence assays showed that the number of TNF-α-positive cells in the lateral ventricles and hippocampus of mice exposed to manganese was increased compared to the control group. After different treatments, the number of TNF-α-positive cells decreased in both the EVs and BDNF@RVG-EVs groups, with a more significant decrease in the BDNF@RVG-EVs group. The CaNa2-EDTA group also showed a decrease, but to a lesser extent (see...). Figure 9 ).

[0061] RT-qPCR results showed that in the lateral ventricle, compared with the manganese group, Tnf-α expression levels decreased: 6.22% in the EVs group, 32.23% in the BDNF@RVG-EVs group, and 11.19% in the CaNa2-EDTA group. Anti-inflammatory factor expression was increased in all groups: Cd206 increased by 22.82% and Arg1 by 52.36% in the EVs group; Cd206 increased by 41.42% and Arg1 by 66.16% in the BDNF@RVG-EVs group; and Cd206 increased by 44.65% and Arg1 by 29.49% in the CaNa2-EDTA group. In the hippocampus, Tnf-α expression decreased: 39.88% in the EVs group, 61.98% in the BDNF@RVG-EVs group, and 47.24% in the CaNa2-EDTA group. The expression of anti-inflammatory factors showed an increasing trend: Cd206 increased by 77.59% and Arg1 increased by 64.32% in the EVs group; Cd206 increased by 80.59% and Arg1 increased by 71.49% in the BDNF@RVG-EVs group; and Cd206 increased by 53.55% and Arg1 increased by 53.79% in the CaNa2-EDTA group (see...). Figure 10 ).

[0062] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of engineered extracellular vesicles in the preparation of products for preventing and treating nerve damage, characterized in that, The engineered extracellular vesicles are extracellular vesicles loaded with brain-derived neurotrophic factor and brain-targeting peptides. The extracellular vesicles are derived from human nasal mucosal mesenchymal stem cells, and the brain-targeting peptide is RVG. 29 peptide, the RVG 29 The amino acid sequence of the peptide is shown in SEQ ID NO.

1.

2. The application of an engineered extracellular vesicle in the preparation of any one of the following products, characterized in that, (1) Promotes neurogenesis; (2) Repairs dopaminergic neurons; (3) Reduces neuroinflammation; The engineered extracellular vesicles are extracellular vesicles loaded with brain-derived neurotrophic factor and brain-targeting peptides, the extracellular vesicles are derived from human nasal mucosal mesenchymal stem cells, and the brain-targeting peptides are RVG. 29 The peptide has the amino acid sequence shown in SEQ ID NO.

1.

3. The application of engineered extracellular vesicles in the preparation of products for treating Parkinson's-like nerve injuries, characterized in that, The engineered extracellular vesicles are extracellular vesicles loaded with brain-derived neurotrophic factor and brain-targeting peptides. The extracellular vesicles are derived from human nasal mucosal mesenchymal stem cells, and the brain-targeting peptide is RVG. 29 The peptide has the amino acid sequence shown in SEQ ID NO.

1.

4. The application according to claim 1, characterized in that, The nerve damage includes nerve damage caused by manganese.

5. The application according to claim 3, characterized in that, The Parkinsonian nerve injury includes Parkinsonian nerve injury caused by manganese exposure.

6. The application according to any one of claims 1 to 3, characterized in that, The product is a nasal preparation.

7. The application according to claim 6, characterized in that, The nasal preparations include nasal drops.

8. The application according to any one of claims 1 to 3, characterized in that, The method for preparing the engineered extracellular vesicles includes the following steps: DSPE-PEG... 2000 -RVG 29 DSPC and cholesterol were dissolved in a methanol-chloroform mixture and rotary evaporated until a film was formed. The film was mixed with a PBS solution containing brain-derived neurotrophic factor (BDNF), sonicated in a water bath, extruded using a filter membrane, and ultrafiltered to obtain a film loaded with BDNF and DSPE-PEG. 2000 -RVG 29 Liposomes were mixed with extracellular vesicles derived from human nasal mucosal mesenchymal stem cells, and after sonication in an ice bath, the mixture was extruded using a filter membrane and ultrafiltered to obtain engineered extracellular vesicles.

9. The application according to claim 8, characterized in that, The method for preparing extracellular vesicles derived from human nasal mucosal mesenchymal stem cells includes the following steps: human nasal polyp tissue is subjected to adherent primary culture to obtain human nasal mucosal mesenchymal stem cells; after 3D dynamic culture of human nasal mucosal mesenchymal stem cells, the supernatant is taken and the extracellular vesicles derived from human nasal mucosal mesenchymal stem cells are separated by differential centrifugation; the differential centrifugation method includes the following steps: the supernatant is centrifuged sequentially at 200~800g for 3~20min, 1000~5000g for 10~60min, 8000~150000g for 30min, and 100000~200000g for 70~120min.

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