Acellular nanovesicle derived from aloe and applications thereof

Abstract

This invention discloses an extracellular nanovesicle derived from *Acorus tatarinowii* and its applications, belonging to the field of biomedical technology. This invention extracts an extracellular nanovesicle from *Acorus tatarinowii* that possesses both anti-neuroinflammatory and blood-brain barrier penetration capabilities. Loading these nanovesicles with siRNA drugs for treating Alzheimer's disease enables efficient delivery of siRNA to the brain lesions, increasing drug concentration in the brain and achieving a synergistic effect. Compared to synthetic nanocarriers, this is more suitable for long-term administration, providing an advantage for the chronic treatment of Alzheimer's disease. The extracellular nanovesicles of this invention can load siRNAs targeting different pathogenic genes, achieving simultaneous intervention on multiple pathogenic links in Alzheimer's disease, and possessing the potential for multi-target synergistic therapy, which is expected to more effectively halt the progression of Alzheimer's disease.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an extracellular nanovesicle derived from Acorus tatarinowii and its applications. Background Technology

[0002] Alzheimer's disease (AD) is a progressive neurodegenerative disease that severely impacts patients' cognition and daily living abilities. With the increasing aging of the global population, the incidence of AD is rising year by year, placing a heavy burden on society and families. Currently, AD treatment faces numerous challenges; existing treatments can only alleviate symptoms and cannot effectively stop disease progression. The blood-brain barrier (BBB), a special structure between blood and brain tissue, effectively blocks most drugs from entering the brain, posing a significant obstacle to drug development for AD. Many drugs with potential therapeutic effects, such as small interfering RNA (siRNA), cannot penetrate the BBB and thus struggle to exert their effects in the brain. Traditional drug delivery systems, such as liposomes and polymer nanoparticles, while improving drug delivery efficiency to some extent, still have limitations in crossing the BBB and achieving brain targeting. They often lack specific brain-targeting capabilities, easily accumulating in non-target tissues, leading to increased side effects. Furthermore, these synthetic carriers may have biocompatibility issues, and long-term use may trigger immune responses. These problems pose significant challenges to current AD drug development and treatment strategies.

[0003] In recent years, plant extravesicles have attracted widespread attention as a novel drug delivery carrier. Plant extravesicles are nanoscale membranous vesicles secreted by plant cells, possessing good biocompatibility, low immunogenicity, and natural targeting capabilities. Therefore, exploring plant-derived extravesicles that can be used for brain-targeted delivery of siRNA for the treatment of Alzheimer's disease (AD) is of great significance for advancing AD treatment strategies. Summary of the Invention

[0004] The purpose of this invention is to provide extracellular nanovesicles derived from *Acorus tatarinowii* and their applications, thereby addressing the problems existing in the prior art. The *Acorus tatarinowii* extracellular nanovesicles provided by this invention can penetrate the blood-brain barrier and possess anti-neuroinflammatory properties. Using them as a carrier for siRNA drugs in the treatment of Alzheimer's disease can efficiently deliver siRNA to the lesion site in the brain, while simultaneously improving the brain microenvironment through anti-neuroinflammatory effects, thus synergistically enhancing the therapeutic effect of AD.

[0005] To achieve the above objectives, the present invention provides the following solution: This invention provides an extracellular nanovesicle from Acorus tatarinowii with brain-targeted delivery capability, the preparation method of which includes the following steps: Take slices of Acorus tatarinowii, add PBS solution, mix and crush, filter, collect the filtrate, centrifuge at differential speed and then at ultracentrifuge, collect the precipitate, resuspend the precipitate and filter through a membrane to extract the extracellular nanovesicles of Acorus tatarinowii. The filter membrane has a pore size of 0.22 μm.

[0006] Furthermore, the mass-to-volume ratio of the Acorus tatarinowii slices to the PBS solution is 1g:10mL.

[0007] Furthermore, the differential centrifugation consists of centrifuging at 1000g for 10 min, centrifuging at 3000g for 30 min, and centrifuging at 10000g for 30 min twice after filtrate.

[0008] Furthermore, the ultracentrifugation is 100,000g × 60 min.

[0009] The present invention also provides the application of the aforementioned Acorus tatarinowii extracellular nanovesicles in the preparation of brain-targeted delivery carriers.

[0010] The present invention also provides a brain-targeted delivery carrier, which is prepared from the extracellular nanovesicles of Acorus tatarinowii.

[0011] The present invention also provides the use of the aforementioned Acorus tatarinowii extracellular nanovesicles as a delivery carrier in the preparation of drugs for treating Alzheimer's disease, Parkinson's disease, glioblastoma or amyotrophic lateral sclerosis.

[0012] Furthermore, the vector can deliver siRNA; The siRNA sequence is shown in SEQ ID NO.4.

[0013] The present invention also provides a drug for treating Alzheimer's disease, Parkinson's disease, glioblastoma or amyotrophic lateral sclerosis, which is obtained by loading the therapeutic drug into the extracellular nanovesicles of Acorus tatarinowii; the therapeutic drug is siRNA, the sequence of which is shown in SEQ ID NO.4.

[0014] The present invention also provides the application of the aforementioned Acorus tatarinowii extracellular nanovesicles in enhancing the ability of drugs to penetrate the blood-brain barrier.

[0015] The present invention discloses the following technical effects: This invention extracts an extracellular nanovesicle from Acorus tatarinowii, which can penetrate the blood-brain barrier. When loaded with siRNA drugs for treating Alzheimer's disease, these siRNAs can be efficiently delivered to the brain lesions, increasing drug concentration and enhancing therapeutic efficacy. Compared to traditional drug delivery systems, this significantly improves brain targeting, reduces distribution in non-target tissues, and lowers side effects.

[0016] The external vesicles provided by this invention have anti-neuroinflammatory effects.

[0017] The external vesicles provided by this invention are derived from natural plants, exhibiting good biocompatibility, low likelihood of triggering immune responses, and high safety. Compared to synthetic nanocarriers, they are more suitable for long-term drug delivery, offering advantages for the chronic treatment of Alzheimer's disease. The external vesicles of this invention can be loaded with siRNAs targeting different pathogenic genes, enabling simultaneous intervention on multiple pathogenic links in Alzheimer's disease, demonstrating the potential for multi-target synergistic therapy and potentially more effectively halting the progression of Alzheimer's disease. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 The images show the particle size and TEM morphology of extracellular vesicles from Acorus tatarinowii. In the images, a represents the particle size and concentration detected by a nanoparticle tracking analyzer (NTA), and b represents the morphology of exosome-like nanovesicles observed by transmission electron microscopy (TEM). Figure 2 The distribution of DiR-labeled ATELNs in mice is shown in the in vivo imaging analysis. Among them, a is in vivo whole-body imaging; b and c are fluorescence images of major ex vivo organs at 8 h and 24 h, respectively; d is in vivo brain imaging; and e is fluorescence image of ex vivo brain tissue at 24 h. Figure 3 The effects of ATLNs on the viability of HMC3 cells (A), SH-SY5Y cells (B), and HCMEC / D3 cells (C); Figure 4 The study aimed to investigate the effects of ATLNs on H2O2-induced oxidative stress damage in SH-SY5Y cells and on the viability of LPS-induced SH-SY5Y cell inflammation models. The study included: A: the effect of H2O2 on SH-SY5Y cell viability; B: the effect of ATLNs on H2O2-induced SH-SY5Y cell viability; C: the effect of LPS on SH-SY5Y cell viability; and D: the effect of conditioned medium on SH-SY5Y cell viability. Figure 5 Fluorescence detection and quantitative statistics of ATLNs in inhibiting LPS-induced oxidative stress in HMC3 cells (a) and (b); Figure 6To assess the cellular uptake and cross-BBB capacity of ATLNs; where A: HMC3 cells; B: SH-SY5Y cells; C: TEER values ​​at 0h and 6h after ATLNs addition; D: efficiency of ATLNs across the in vitro BBB. Figure 7 Representative swimming trajectories of ATELNs mice during the MWM test training and space exploration phases; Figure 8 Images showing the appearance of blank gel (A) and AELNs-gel gel (B) before and after gelation; Figure 9 Dynamic strain scans of blank gel (A) and AELNs-gel gel (B); Figure 10 Dynamic frequency scans of blank gel (A) and AELNs-gel gel (B); Figure 11 Dynamic temperature scans of blank gel (A) and AELNs-gel gel (B); Figure 12 Mechanical stability of ATELNs-gel (A) and particle size variation at different storage times (B); Figure 13 The following are representative swimming trajectories of ATELNs-gel group mice during the MWM test training and spatial exploration phases; where A represents representative swimming trajectories of different groups of mice during the water maze test training and spatial exploration phases; B represents escape latency; C represents swimming speed; D represents the number of times the platform was crossed; E represents the time spent in the target quadrant; and F represents the distance traveled in the target quadrant. (n=7, compared with the control group, ## P <0.01; compared with the LPS group, P <0.05, P <0.01); Figure 14 H&E staining was performed on the hippocampus (DG, CA1, CA3) and cortical region of mice in each group; Figure 15 Iba-1 immunofluorescence staining was performed on the hippocampus (DG, CA1, CA3) and cortical region of mice in each group. Figure 16 Immunofluorescence staining of GFAP in the hippocampus (DG, CA1, CA3) and cortical region of mice in each group was performed. Figure 17 iNOS immunofluorescence staining was performed on the hippocampus (DG, CA1, CA3) and cortical regions of mice in each group. Detailed Implementation

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0023] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0024] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0025] Example 1: Extraction and Characterization of Exovesicles from Acorus tatarinowii 1. Extraction of exovesicles from Acorus tatarinowii Extravesicles were extracted from *Acorus tatarinowii* medicinal material using differential centrifugation. The specific steps were as follows: The *Acorus tatarinowii* medicinal material was washed three times with pure water. It was then mixed with PBS buffer (pH=7.4) at a material-to-liquid ratio of 1:10 (g:mL) at 4 ℃. The mixture was placed in a high-speed blender and blended for 1 minute followed by a 1-minute pause, repeated 5 times. After filtration through medical gauze, the filtrate was collected and aliquoted into centrifuge tubes. The tubes were centrifuged twice, once at 1000 g for 10 min, once at 3000 g for 30 min, and once at 10000 g for 30 min. The filtrate was then filtered through a 0.45 μm filter membrane. Next, the filtrate was centrifuged at 100000 g for 60 min using an ultracentrifuge. The supernatant was removed, the precipitate was resuspended in PBS and vortexed for 1 min, then centrifuged at 10000 g for 15 min. The supernatant was then filtered through a 0.22 μm filter membrane to obtain ATELNs. All operations were performed at 4 °C, and the extracted samples were immediately stored in a -80 °C refrigerator.

[0026] 2. Characterization of the external vesicles of Acorus tatarinowii Characterization results as follows Figure 1 Nanoparticle tracking and analysis (NTA) was used to detect exosome-like nanovesicles from *Acorus calamus*, and the results are as follows: Figure 1 As shown in figure a, the average particle size of the outer vesicles is concentrated at 140.9 nm, with a concentration of approximately 2.9 × 10⁻⁶. 11 Particles / mL.

[0027] Transmission electron microscopy (TEM) was used to observe the morphology of exosome-like nanovesicles from Acorus tatarinowii. The nanovesicles were found to exhibit a nearly circular, disc-shaped structure with a dark center and translucent edges, exhibiting typical vesicle-like structure. Figure 1 (b).

[0028] Example 2: Extracellular nanovesicles of Acorus tatarinowii penetrating the blood-brain barrier and efficacy verification 1. Brain-targeted research The extracellular nanovesicles of Acorus tatarinowii prepared in Example 1 were labeled with DiR using an incubation method. Twelve healthy C57BL / 6 mice with a weight range of 18-20 g were selected and randomly divided into a free DiR group and a DiR-labeled extracellular nanovesicle group. The mice were administered the drugs via tail vein injection, and their behavior, weight and other physiological indicators were observed regularly.

[0029] At time points of 1 h, 2 h, 4 h, 6 h, 12 h, 24 h, and 48 h after drug administration, mice were placed in a small animal in vivo imaging system and anesthetized with isoflurane. Whole-body fluorescence images of the mice were acquired to preliminarily observe the distribution of labeled extracellular nanovesicles in the mice, with a focus on the fluorescence signal intensity in the brain, and the fluorescence intensity values ​​were recorded. After imaging observation, the mice were sacrificed, and tissues such as the brain, heart, liver, spleen, lungs, and kidneys were harvested. These tissues were rinsed with physiological saline to remove surface blood and impurities, blotted dry with filter paper, weighed using an electronic balance, and the weight recorded. The tissues were then observed in a small animal in vivo imaging system. The results are as follows: Figure 2 As shown, extracellular nanovesicles (ATELNs) of Acorus tatarinowii exhibit significant brain targeting, but their efficiency in penetrating the BBB is insufficient.

[0030] 2. Verification of the anti-neuroinflammatory efficacy of extracellular nanovesicles of Acorus tatarinowii This invention systematically analyzes the anti-neuroinflammatory and neuroprotective effects of ATLNs at both the cellular and animal levels. At the cellular level, the toxicity of ATLNs to SH-SY5Y, HMC3, and HCMEC / D3 cells was first detected using the CCK-8 assay to determine the appropriate dosage. Figure 3 Furthermore, using an H2O2-induced SH-SY5Y cell oxidative stress model and an LPS-induced HMC3 cell inflammation model, the neuroprotective, anti-inflammatory, and ROS-improving effects of ATLNs were investigated. Figure 4 Simultaneously, the uptake of ATEELNs by cells was observed, and their in vitro BBB crossing rate was detected. Figure 6 Furthermore, it was confirmed that ATELNs can dose-dependently reduce LPS-induced ROS production in HMC3 cells, with 100 μg / mL showing the best effect. Figure 5 (a) Transwell assays clearly show that 6 hours is the optimal time point for detecting cell migration dynamics. Figure 5 (b) Animal studies used C57BL / 6 mice as the research subjects. A neuroinflammatory model was established by intraperitoneal injection of lipopolysaccharide (LPS). The effect of Acorus tatarinowii external vesicles (ATELNs) on the learning and memory function of the model mice was evaluated by the Morris water maze (MWM) test. Figure 7 ).

[0031] Example 3 Hydrogel-loaded Acorus calamus exosomes 1. Formulation screening of hydrogel-loaded Acorus tatarinowii exosomes (ATELNs-gel) A blank thermosensitive gel was prepared by adding the prescribed amounts of P407, P188, and HMPC to pre-cooled PBS (pH=7.4) using the cold dissolution method, stirring and dissolving in an ice bath, and then standing overnight at 4°C. The gelation temperature was determined by the inverted method. The effects of different mass concentrations of P407, P188, and HPMC on the gelation temperature were investigated by single-factor experiments. The formulation was then optimized and validated using Box-Behnken design with Design-Expert 10.0 software. Finally, the ultrafiltration concentrated AELNs were diluted to 1 mg / mL, and the three materials were added in the optimal amounts. The mixture was stirred and dissolved in an ice bath and then stood overnight at 4°C to obtain the AELNs-gel.

[0032] 2. Evaluation of hydrogel formulations ① Appearance of the gel: The appearance of the gel at room temperature, its gelation state at 35℃, and its rewarming morphology were examined and photographed. It was found that the blank gel was a colorless, clear, and transparent liquid at room temperature, and after gelation, it became a colorless, transparent, semi-solid gel. The ATLNs-gel was a brownish-yellow, free-flowing liquid at room temperature, and after gelation, it became a brownish-yellow, semi-solid gel. Figure 8 ( ), the phase transition is reversible.

[0033] ② pH value and gelation time determination: The loading of ATLNs did not significantly change the gelation temperature, gelation time and pH of the gel, indicating that ATLNs have good compatibility with the temperature-sensitive matrix and meet the requirements for in situ nasal drug delivery, as shown in Table 1.

[0034] Table 1. Gelation temperature, gelation time, and pH value (n=3) ③ Rheological Study: The viscoelastic properties of AELNs-gel were investigated using a rheometer. Dynamic strain scanning showed that the storage modulus G' plateau value of AELNs-gel (9964 Pa) was higher than that of the blank gel (8319 Pa), the linear viscoelastic region was wider, and the critical yield stress was greater, indicating that the addition of AELNs significantly enhanced the structural stability and shear resistance of the gel. Dynamic frequency scanning showed that G' was always greater than G'', indicating that the gel elasticity was dominant and the structure was stable. The loss modulus G" of the gel decreased with increasing shear frequency, indicating that it is a non-Newtonian fluid with shear-thinning characteristics. Dynamic temperature scanning determined the phase transition temperature of AELNs-gel to be 31.16℃, consistent with the results of the inverted method. Figure 9-11 ).

[0035] ④ Stability Study: Mechanical stability tests showed that AELNs-gel exhibited good mechanical stability after centrifugation at 10000 g for 30 min without stratification or precipitation. Storage stability studies indicated that AELNs-gel maintained stable particle size for 7 days at 4℃ in the dark; however, the particle size increased after 7 days. Figure 12 ).

[0036] 3. Validation of the efficacy of hydrogel-loaded Acorus tatarinowii exosomes (animal experiment) In a mouse model of LPS-induced neuroinflammation, Morris water maze results showed that ATLNs and ATLNs-gel effectively shortened the escape latency, increased the number of platform crossings and the proportion of activity in the target quadrant, and significantly improved LPS-induced learning and cognitive impairment, with the gel formulation showing better results. Figure 13 HE staining of brain tissue showed that pathological damage to hippocampal and cortical neurons was significantly alleviated after drug administration. Figure 14 Immunofluorescence results confirmed that ATELNs and the composite gel could inhibit excessive activation of glial cells, downregulate iNOS expression, and suppress intracranial inflammatory responses. Figure 15-17 In summary, ATELNs can reduce neuroinflammation, protect neurons, and improve cognitive function, while the thermosensitive gel carrier can further enhance the therapeutic effect in vivo.

[0037] Example 4: Design and Validation of AD siRNA 1. siRNA sequence design Based on the pathogenesis of Alzheimer's disease (AD), siRNA sequences targeting the key pathogenic gene BACE1 were selected. Homo sapiens APP and Homo sapiens BACE1 were retrieved from the National Center for Biotechnology database. Transcripts of the APP and BACE1 genes were selected based on their mRNA sequences, exported, and siRNA sequences were designed using online design software. Sequences with the highest scores and whose first and second bp were G or C were selected (Table 2). Among them, the siRNA-4 group showed the lowest BACE1 expression level, with a silencing efficiency of 82.05%. siRNA-4 exhibited the highest silencing effect and was used as the nucleic acid drug in subsequent experiments. A negative control siRNA sequence was also designed, with a base sequence that showed no homology to the target gene.

[0038] Table 2. Sequence design of siBACE1 2. siRNA sequence modification To improve the stability of siRNA and its binding efficiency to plant exovesicles, methoxylation was performed. The 2'-O-methylation method was used to modify the nucleotides at positions 2, 4, and 6 of both the sense and antisense strands of the siRNA.

[0039] 3. Loading and evaluation of external vesicles and AD siRNA ① The purified exosomes and AD siRNA were thoroughly mixed in PBS buffer at a certain mass ratio and placed in an electroporation cuvette for electroporation. Then, the mixture was incubated at 37 °C for 4 h to allow AD siRNA to bind to the surface of the exosomes or enter the vesicles through electrostatic adsorption and membrane permeation, thus completing the loading of AD siRNA and obtaining the AD siRNA@ATELNs complex.

[0040] ② The particle size and potential of the AD siRNA@ATELNs complex were determined using NTA, and the microstructure of the complex was observed using transmission electron microscopy (TEM). The formation of the AD siRNA@Ac-EVs complex was verified by agarose gel electrophoresis, and its drug loading and encapsulation efficiency were determined using SYBR Gold staining combined with Image J software. The stability of the AD siRNA complex in storage and serum were investigated. The results showed that AD siRNA could be effectively loaded into the exosomes of Acorus tatarinowii while maintaining its intact morphology and good stability.

[0041] 3. Evaluation of the anti-AD effect of extracellular vesicles loaded with AD siRNA This invention analyzes the anti-AD therapeutic effect of siRNA-loaded extracellular vesicles at both cellular and animal levels. Using H2O2-induced SH-SY5Y cell oxidative stress models and LPS-induced HMC3 cell inflammation models as research subjects, the study investigated cell viability, apoptosis levels, expression of AD-related pathogenic proteins, and inflammatory factor levels. The results showed that siRNA-loaded vesicles protected against cell damage and significantly improved AD pathological features. At the animal level, using AD model mice as research subjects, behavioral experiments such as the water maze and novel object recognition were conducted to assess the mice's learning, memory, and cognitive functions. Combined with pathological staining, quantification of pathogenic proteins, and detection of inflammation and synapse-related indicators, the results confirmed that AD-loaded siRNA-loaded extracellular vesicles had a certain alleviating effect on pathological brain damage in mice and could improve cognitive function.

[0042] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An extracellular nanovesicle from Acorus tatarinowii with brain-targeted delivery capability, characterized in that, The preparation method includes the following steps: Take slices of Acorus tatarinowii, add PBS solution, mix and crush, filter, collect the filtrate, centrifuge at differential speed and then at ultracentrifuge, collect the precipitate, resuspend the precipitate and filter through a membrane to extract the extracellular nanovesicles of Acorus tatarinowii. The filter membrane has a pore size of 0.22 μm.

2. The extracellular nanovesicles of Acorus tatarinowii according to claim 1, characterized in that, The mass-to-volume ratio of the Acorus tatarinowii slices to the PBS solution is 1g:10mL.

3. The extracellular nanovesicles of Acorus tatarinowii according to claim 1, characterized in that, The differential centrifugation consisted of centrifuging at 1000g for 10 min, centrifuging at 3000g for 30 min, and centrifuging at 10000g for 30 min twice after filtrate.

4. The extracellular nanovesicles of Acorus tatarinowii according to claim 1, characterized in that, The ultracentrifugation was performed at 100,000g for 60 minutes.

5. The application of the extracellular nanovesicles of Acorus tatarinowii as described in any one of claims 1 to 4 in the preparation of brain-targeted delivery carriers.

6. A brain-targeting delivery vector, characterized in that, It is prepared from the extracellular nanovesicles of Acorus tatarinowii as described in any one of claims 1 to 4.

7. The use of the extracellular nanovesicles of Acorus tatarinowii as described in any one of claims 1 to 4 as a delivery carrier in the preparation of medicaments for treating Alzheimer's disease, Parkinson's disease, glioblastoma or amyotrophic lateral sclerosis.

8. The application according to claim 7, characterized in that, The vector delivers siRNA; The sequence of the siRNA is shown in SEQ ID NO.

4.

9. A drug for treating Alzheimer's disease, Parkinson's disease, glioblastoma, or amyotrophic lateral sclerosis, characterized in that, The therapeutic drug is obtained by loading extracellular nanovesicles of Acorus tatarinowii according to any one of claims 1 to 4; the therapeutic drug is siRNA, the sequence of which is shown in SEQ ID NO.

4.

10. The use of the extracellular nanovesicles of Acorus tatarinowii as described in any one of claims 1 to 4 in enhancing the ability of drugs to penetrate the blood-brain barrier.

Images