Intranasal microneedle drug delivery system for Alzheimer's disease and preparation method thereof

Abstract

The invention discloses an Alzheimer's disease transnasal microneedle drug delivery system and a preparation method thereof, and relates to the technical field of biological medicine, the microneedle drug delivery system comprises a microneedle substrate and a microneedle tip; the microneedle tip comprises a medicine carrying exosome, hyaluronic acid and polyvinylpyrrolidone; the drug-loaded exosome comprises a tobacco leaf exosome, the surface of the tobacco leaf exosome is connected with brain-targeted peptide RVG29, and donepezil is wrapped in the tobacco leaf exosome. The invention further discloses a preparation method of the microneedle drug delivery system. According to the nasal microneedle drug delivery system for Alzheimer's disease, the delivery efficiency of a small molecule drug donepezil to the brain is enhanced, and the nasal microneedle drug delivery system has the advantages of being high in safety, good in curative effect and good in targeting property; the problems that in the prior art, small molecule medicine donepezil difficultly passes through the blood brain barrier and enters the central nervous system, the medicine delivery efficiency is low, the treatment effect is low, and side effects are many are solved.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceutical technology, and particularly to a transnasal microneedle drug delivery system for Alzheimer's disease and a preparation method thereof. Background Art

[0002] Alzheimer's disease (AD) is a neurodegenerative disease of the central nervous system. It is highly prevalent in the middle-aged and elderly population, manifested as memory impairment, cognitive dysfunction, decline in activities of daily living, etc. The pathogenesis of Alzheimer's disease is complex and related to multiple factors such as genetic genes, lifestyle, and environment. Donepezil hydrochloride (DPH) is one of the commonly used oral drugs for the treatment of Alzheimer's disease in clinical practice. Due to its dosage form and administration method, the amount of donepezil entering the central nervous system is small, resulting in side effects such as fatigue, nausea, vomiting, and insomnia, and leading to low therapeutic effects. Therefore, it is urgent to develop a drug preparation that can efficiently penetrate the blood-brain barrier and enter the central nervous system for the treatment of Alzheimer's disease.

[0003] Transnasal drug delivery can penetrate the blood-brain barrier, and the drug delivery efficiency into the central nervous system is significantly enhanced compared with oral administration. The transnasal drug delivery methods include: nasal sprays, nasal inhalants, nasal drops, transdermal patches, etc. Microneedles (MN) belong to one of the transdermal patches. Compared with the traditional transnasal drug delivery system, the microneedle drug delivery system can be pasted inside the nasal cavity, penetrate the stratum corneum directionally, and directly deliver the drug to the epidermis or dermis layer in a minimally invasive form to exert its drug effect. In addition, while microneedles exert targeted drug delivery, the trauma to the body is also reduced. Therefore, how to design and prepare a drug delivery system with microneedles loaded with donepezil and targeted to the brain, reducing the trauma to the body and side effects, is an effective way to further improve the treatment effect of Alzheimer's disease. Summary of the Invention

[0004] In order to solve the above technical problems, the purpose of the present invention is to provide a transnasal microneedle drug delivery system for Alzheimer's disease and a preparation method thereof. The transnasal microneedle drug delivery system for Alzheimer's disease of the present invention enhances the delivery efficiency of the small molecule drug donepezil to the brain, and has the advantages of high safety, good efficacy, and good targeting, solving the problems in the prior art such as the difficulty of the small molecule drug donepezil in passing through the blood-brain barrier, low drug delivery efficiency into the central nervous system, low therapeutic efficacy, and many side effects.

[0005] The technical solution of the present invention to solve the above technical problems is as follows: providing a transnasal microneedle drug delivery system for Alzheimer's disease, including a microneedle base and a microneedle tip; the microneedle tip includes a drug-loaded exosome, hyaluronic acid, and polyvinylpyrrolidone;

[0006] The drug-loaded exosomes include tobacco leaf exosomes, the surface of which is conjugated with the brain-targeting peptide RVG29, and donepezil is encapsulated inside the tobacco leaf exosomes.

[0007] Furthermore, the microneedle base is polyvinylpyrrolidone.

[0008] Furthermore, the tobacco leaf exosomes are prepared by the following method: mixing tobacco leaves with water for juicing, then performing gradient centrifugation, and further purifying by sucrose density gradient centrifugation to obtain tobacco leaf exosomes.

[0009] Still further, the gradient centrifugation is successively: centrifuging at 8000g for 45 min, 10000g for 1 h, 12000g for 2 h, and 120000g for 12 h.

[0010] Still further, centrifuging at 150000g for 12 h in 8%, 30%, 45%, and 60% sucrose solutions from top to bottom. The centrifugation temperature is 4°C.

[0011] The present invention also provides a preparation method of the above nasal microneedle drug delivery system for Alzheimer's disease, comprising the following steps:

[0012] (1) Mixing the brain-targeting peptide RVG29 and tobacco leaf exosomes, and performing ultrasonic treatment to obtain modified exosomes;

[0013] (2) Mixing donepezil with the modified exosomes obtained in step (1), incubating, and performing ultrafiltration to obtain drug-loaded exosomes;

[0014] (3) Uniformly mixing hyaluronic acid, polyvinylpyrrolidone, and the drug-loaded exosomes obtained in step (2), adding them into a microneedle mold, centrifuging, and drying and curing to obtain the microneedle tip;

[0015] (4) Adding polyvinylpyrrolidone to the bottom of the microneedle tip obtained in step (3), centrifuging, and drying and curing to obtain the nasal microneedle drug delivery system for Alzheimer's disease.

[0016] Furthermore, in step (1), the volume ratio of the brain-targeting peptide RVG29 to tobacco leaf exosomes is 400 - 600 μL: 400 - 600 μL; the concentration of tobacco leaf exosomes is 6.10×10 11 particles / mL.

[0017] Furthermore, in step (2), incubating at a temperature of 35 - 40°C for 3 - 5 h.

[0018] Furthermore, in step (2), ultrafiltration is performed using an ultrafiltration tube with Mw = 3 kDa.

[0019] Furthermore, in step (2), the volume ratio of donepezil to modified exosomes is 1 - 2 mL: 400 - 600 μL.

[0020] Further, in step (3), the mass-volume ratio of hyaluronic acid, polyvinylpyrrolidone and drug-loaded exosomes is 0.1 - 0.3 g : 0.1 g : 0.8 - 1.2 mL.

[0021] Further, in step (3), centrifuge at 3500 rpm for 15 min.

[0022] Further, in step (3), dry at 37 °C for 30 min and repeat twice.

[0023] Further, in step (4), centrifuge at 3500 rpm for 3 min.

[0024] Further, in step (4), dry at 37 °C until it solidifies and forms a shape.

[0025] The present invention has the following beneficial effects:

[0026] 1. The present invention uses tobacco leaf exosomes as a drug delivery carrier, which has significant advantages such as low immunogenicity, high stability, high loading capacity and targeting. As a carrier for loading the small molecule drug donepezil, tobacco leaf exosomes can improve the drug targeting and enhance its delivery efficiency into the central nervous system. Moreover, the results of lipid component analysis prove that lipid components such as hexosylceramide, sphingolipids and phosphatidylcholine contained in tobacco leaf exosomes have potential nerve repair and protection effects. When combined with donepezil for the treatment of Alzheimer's disease, the treatment effect can be improved.

[0027] 2. The present invention extracts exosomes from tobacco leaves and performs surface modification by connecting the brain-targeting peptide RVG29, and then loads the small molecule drug donepezil for the treatment of Alzheimer's disease inside the modified exosomes. The connection of the brain-targeting peptide endows it with active targeting, reducing the side effects caused by the systemic exposure of the drug. The lipid bilayer structure of exosomes can not only provide a relatively stable environment for the encapsulated small molecule drug donepezil, but also has a certain ability to cross biological barriers, making the drug-loaded exosomes more likely to cross the blood-brain barrier and achieve more precise and effective treatment of Alzheimer's disease.

[0028] 3. The present invention uses microneedles as a drug delivery system and adopts the method of nasal administration, which synergistically reduces the trauma to the body while the drug-loaded exosomes are easy to cross the blood-brain barrier, thereby significantly enhancing the drug delivery efficiency into the central nervous system and providing new ideas for the treatment of Alzheimer's disease and other brain diseases. Description of the Drawings

[0029] Figure 1 It is a transmission electron microscope image of the tobacco leaf exosomes extracted in Example 1;

[0030] Figure 2High performance liquid chromatography chart of tobacco leaf exosomes extracted in Example 1;

[0031] Figure 3 Lipid component analysis chart of tobacco leaf exosomes extracted in Example 1;

[0032] Figure 4 Ultraviolet-visible spectroscopy chart of drug-loaded exosomes prepared in Example 1;

[0033] Figure 5 Digital picture of the intranasal microneedle drug delivery system for Alzheimer's disease prepared in Example 1;

[0034] Figure 6 Solubility characterization result chart of the intranasal microneedle drug delivery system for Alzheimer's disease prepared in Example 1;

[0035] Figure 7 DID fluorescence intensity comparison chart in the brains of AD rats;

[0036] Figure 8 Volcano plot of differential genes between the model group and the treatment group;

[0037] Figure 9 GO enrichment analysis chart of differential genes;

[0038] Figure 10 Cluster analysis heat map of differential genes;

[0039] Figure 11 KEGG enrichment scatter plot of differential genes. Detailed implementation manners

[0040] The principles and characteristics of the present invention are described below. The examples given are only used to explain the present invention and are not intended to limit the scope of the present invention. Those not specified in the examples are carried out under conventional conditions or conditions recommended by the manufacturer. Those reagents or instruments not specified by the manufacturer can be obtained as conventional products through commercial purchase.

[0041] Example 1

[0042] An intranasal microneedle drug delivery system for Alzheimer's disease, comprising a microneedle base and a microneedle tip; the microneedle tip comprises drug-loaded exosomes, hyaluronic acid and polyvinylpyrrolidone; the drug-loaded exosomes comprise tobacco leaf exosomes, a brain-targeting peptide RVG29 is connected to the surface of the tobacco leaf exosomes, and donepezil is encapsulated inside the tobacco leaf exosomes; the microneedle base is polyvinylpyrrolidone;

[0043] Among them, the tobacco leaf exosomes are prepared by the following method: Mix tobacco leaves and water in a ratio of 240 g: 120 mL, juice and filter, then centrifuge at 8000 g for 45 min, 10000 g for 1 h, 12000 g for 2 h, and 120000 g for 12 h in sequence. Then add sucrose solutions with concentrations of 8%, 30%, 45%, and 60% from top to bottom, and centrifuge at 4 °C and 150000 g for 12 h. Finally, resuspend in PBS solution to obtain tobacco leaf exosomes;

[0044] The preparation method of the intranasal micro-needle drug delivery system for Alzheimer's disease includes the following steps:

[0045] (1) Mix the brain-targeting peptide RVG29 and 6.10×10 11 particles / mL tobacco leaf exosomes in a volume ratio of 500 μL: 500 μL, and perform ultrasonic treatment to obtain modified exosomes;

[0046] (2) Mix donepezil and the modified exosomes obtained in step (1) in a volume ratio of 1.5 mL: 500 μL, incubate at 37 °C for 4 h, and perform ultrafiltration using an ultrafiltration tube with Mw = 3 kDa to obtain drug-loaded exosomes;

[0047] (3) Mix hyaluronic acid, polyvinylpyrrolidone, and the drug-loaded exosomes obtained in step (2) in a mass-volume ratio of 0.2 g: 0.1 g: 1 mL, mix evenly, add to a micro-needle mold, centrifuge at 3500 rpm for 15 min, remove all air bubbles, dry and cure at 37 °C for 30 min, and repeat twice to obtain the micro-needle tip;

[0048] (4) Add polyvinylpyrrolidone to the bottom of the micro-needle tip obtained in step (3), centrifuge at 3500 rpm for 3 min, remove all air bubbles, dry at 37 °C, and wait for it to solidify and form to obtain the intranasal micro-needle drug delivery system for Alzheimer's disease.

[0049] Example 2

[0050] An intranasal micro-needle drug delivery system for Alzheimer's disease, comprising a micro-needle base and a micro-needle tip; the micro-needle tip includes drug-loaded exosomes, hyaluronic acid, and polyvinylpyrrolidone; the drug-loaded exosomes include tobacco leaf exosomes, the surface of the tobacco leaf exosomes is connected with the brain-targeting peptide RVG29, and donepezil is encapsulated inside the tobacco leaf exosomes; the micro-needle base is polyvinylpyrrolidone;

[0051] Its preparation method includes the following steps:

[0052] (1) Mix the brain-targeting peptide RVG29 and 6.10×10 11 particles / mL tobacco leaf exosomes in a volume ratio of 400 μL: 600 μL, and perform ultrasonic treatment to obtain modified exosomes;

[0053] (2) Mix donepezil with the modified exosomes obtained in step (1) at a volume ratio of 1 mL: 400 μL, incubate at 35 °C for 3 h, and perform ultrafiltration using an ultrafiltration tube with Mw = 3 kDa to obtain drug-loaded exosomes;

[0054] (3) Mix hyaluronic acid, polyvinylpyrrolidone, and the drug-loaded exosomes obtained in step (2) at a mass-volume ratio of 0.1 g: 0.1 g: 0.8 mL, add them to a microneedle mold, centrifuge at 3500 rpm for 15 min to remove all air bubbles, and dry and cure at 35 °C for 20 min to obtain microneedle tips;

[0055] (4) Add polyvinylpyrrolidone to the bottom of the microneedle tips obtained in step (3), centrifuge at 3500 rpm for 3 min to remove all air bubbles, dry at 35 °C, and wait for it to solidify and form to obtain a transnasal microneedle drug delivery system for Alzheimer's disease.

[0056] Example 3

[0057] A transnasal microneedle drug delivery system for Alzheimer's disease, comprising a microneedle base and microneedle tips; the microneedle tips include drug-loaded exosomes, hyaluronic acid, and polyvinylpyrrolidone; the drug-loaded exosomes include tobacco leaf exosomes, and the surface of the tobacco leaf exosomes is connected with the brain-targeting peptide RVG29, and donepezil is encapsulated inside the tobacco leaf exosomes; the microneedle base is polyvinylpyrrolidone;

[0058] Its preparation method includes the following steps:

[0059] (1) Mix the brain-targeting peptide RVG29 and 6.10×10 11 particles / mL tobacco leaf exosomes at a volume ratio of 600 μL: 400 μL, and perform ultrasonic treatment to obtain modified exosomes;

[0060] (2) Mix donepezil with the modified exosomes obtained in step (1) at a volume ratio of 2 mL: 600 μL, incubate at 40 °C for 5 h, and perform ultrafiltration using an ultrafiltration tube with Mw = 3 kDa to obtain drug-loaded exosomes;

[0061] (3) Mix hyaluronic acid, polyvinylpyrrolidone, and the drug-loaded exosomes obtained in step (2) at a mass-volume ratio of 0.3 g: 0.1 g: 1.2 mL, add them to a microneedle mold, centrifuge at 3500 rpm for 15 min to remove all air bubbles, and dry and cure at 40 °C for 40 min to obtain microneedle tips;

[0062] (4) Add polyvinylpyrrolidone to the bottom of the microneedle tips obtained in step (3), centrifuge at 3500 rpm for 3 min to remove all air bubbles, dry at 40 °C, and wait for it to solidify and form to obtain a transnasal microneedle drug delivery system for Alzheimer's disease.

[0063] Test Example 1 Observation of the morphological characteristics of tobacco leaf exosomes by transmission electron microscopy

[0064] Take 10 μL of the tobacco leaf exosomes extracted in Example 1 and place them on the surface of a copper grid. After 10 min, use filter paper to absorb the excess liquid. Take 10 μL of ultrapure water and drop it on the copper grid. After 30 s, use filter paper to absorb the excess liquid. Then add 10 μL of 3% uranyl acetate. After the sample has fully absorbed, use filter paper to absorb the excess uranyl acetate. After drying, place it in a transmission electron microscope to observe the morphological characteristics. The results of the transmission electron microscope are as Figure 1 shown.

[0065] Test Example 2 Analysis of nicotine in tobacco leaf exosomes by high performance liquid chromatography

[0066] Take 500 μL of the tobacco leaf exosomes extracted in Example 1 and place them in a 5 mL volumetric flask. Add an appropriate amount of methanol, sonicate for 30 min, then add methanol to make up the volume to 5 mL. Then filter through a 0.22 μm microporous filter membrane to obtain the test solution. Take 250 μL of nicotine (10 mg / mL) and place it in a 5 mL volumetric flask. Add methanol to dissolve and make up the volume. Filter through a 0.22 μm microporous filter membrane to obtain the nicotine reference substance. Use a Waters C18 chromatographic column (150 mm × 4.6 mm, 5 μm) for nicotine chromatographic determination. The injection volume is 20 μL, and the mobile phase consists of methanol (A) and 0.02 mol / L KH2SO4 (pH = 6) (B). The flow rate is 0.6 mL / min, and the wavelength is 260 nm. The isocratic program is as follows: A:B = 40:60, and the column temperature is 30 °C.

[0067] The results of the analysis of nicotine in tobacco leaf exosomes by high performance liquid chromatography are as Figure 2 shown. The peak area of nicotine is 11935336, the peak area of tobacco leaf exosomes is 13158322, the concentration of the nicotine reference substance is 0.5 mg / mL, and the calculated nicotine concentration in tobacco leaf exosomes is 0.45 mg / mL. The analysis results show that tobacco leaf exosomes are rich in nicotine.

[0068] Test Example 3 Analysis of the lipid components of tobacco leaf exosomes

[0069] Perform lipid component analysis on the tobacco leaf exosomes extracted in Example 1. The analysis results are as Figure 3 shown. The results show that it contains components such as hexosylceramide, sphingolipids, and phosphatidylcholine, which are used for the treatment of Alzheimer's disease.

[0070] Test Example 4 UV-visible spectral characterization of drug-loaded exosomes

[0071] Place the tobacco leaf exosomes, donepezil, and drug-loaded exosomes prepared in Example 1 in a 96-well plate respectively. Use an enzyme-linked immunosorbent assay instrument to perform wavelength scanning after calibration at 300 - 700 nm. The results are as Figure 4 shown, which proves the successful assembly.

[0072] Experimental Example 5 Morphological Characterization of Transnasal Microneedle Drug Delivery System for Alzheimer's Disease

[0073] The digital picture of the transnasal microneedle drug delivery system for Alzheimer's disease prepared in Example 1 is as Figure 5 shown. The image can clearly show that the microneedles are composed of 400 (20×20) microneedles on the bottom plate. The needle height is about 300 μm, the base diameter of each needle cavity is 160 μm, the center distance between adjacent needles is 550 μm, the microneedles are conical in shape, with uniform size and sharp tips, which are necessary for inserting into the skin.

[0074] Experimental Example 6 Dissolution Performance of Transnasal Microneedle Drug Delivery System for Alzheimer's Disease

[0075] To evaluate the dissolution performance of the microneedles after inserting into the skin, 1.4% agarose gel was used to simulate the skin. After the transnasal microneedle drug delivery system for Alzheimer's disease prepared in Example 1 was inserted into the agarose gel, the morphology of the microneedles at different time points was observed through a microscope to evaluate the dissolution effect of the microneedles in each time period. The results are as Figure 6 shown.

[0076] Experimental Example 7 Detection of DID Fluorescence Intensity in the Brains of AD Rats by Small Animal In Vivo Imaging System

[0077] (1) Nine AD model rats were taken and randomly divided into 3 groups, with 3 rats in each group. They were fasted for 12 h before the formal experiment and allowed free access to water.

[0078] (2) The rats were anesthetized.

[0079] (3) Appropriate amounts of microneedles were cut, and DID dye (Ⅰ), exosomes labeled with DID dye (Ⅱ)

[0080] and microneedles labeled with DID dye (Ⅲ) were pasted inside the nasal cavities of the rats respectively.

[0081] (4) The rats were dissected at 0.5 h, 1 h, and 1.5 h after drug administration, and the small animal in vivo imaging system was used to collect, record, and compare the distribution of fluorescence intensity in the brain tissues of each group of rats. The results are as Figure 7 shown. The results show that the microneedle drug delivery system prepared in this Experimental Example 1 has stronger fluorescence intensity, indicating that its delivery effect to the brain tissue is better. This method provides new ideas for the treatment of Alzheimer's disease and further application in the treatment of other brain diseases.

[0082] Experimental Example 7 Transcriptome Analysis

[0083] To further study the treatment mechanism of the transnasal microneedle drug delivery system for Alzheimer's disease on AD rats, differential comparison was carried out on the hippocampal tissues of the brains of the model group and the treatment group through transcriptome analysis.

[0084] (1) Set the significance P-value < 0.05 and the fold change |log2FC| > 1, and perform differential expression analysis on all genes in the model group and the treatment group. The volcano plot of differentially expressed genes is as shown in Figure 8 . The results show that a total of 161 genes exhibited significant transcriptional differences, and these genes are called differentially expressed genes (DEGs). Among them, 44 genes were significantly up-regulated (red dots), while 117 genes were significantly down-regulated (blue dots).

[0085] (2) The GO enrichment analysis diagram of differentially expressed genes is as shown in Figure 9 . Among them, the differentially expressed genes are divided into three parts: molecular function (MF), biological process (BP), and cellular component (CC). The results show that compared with the model group, the enriched pathways of differentially expressed genes are mainly concentrated in cellular processes, biological regulation, response to stimuli, regulation of biological processes, protein-containing complexes, binding, and catalytic activity, etc. These results provide important clues for understanding the impact of the treatment group on gene expression changes.

[0086] (3) The heatmap of cluster analysis of differentially expressed genes is as shown in Figure 10 . The results show that compared with the model group, genes such as Chrna5, Slc17a6, Ddc, Mc5r, Nxph3, Clspn, and Prokr1 were significantly up-regulated. These genes play important roles in pathways such as neural signal transduction, neurotransmitter synthesis and metabolism, and inflammatory responses, and are closely related to nervous system diseases such as AD, suggesting that these genes may be potential biomarkers or therapeutic targets. Among them, Chrna5 is one of the subunits of the nicotinic acetylcholine receptor and is involved in acetylcholine-mediated signal transduction. The significantly down-regulated genes mainly include Oas1a, Tap1, RT1-M1-4, Oas1i, Mefv, C1qtnf3, RT1-CE1, Mnda, Tnfrsf14, Twist1, Dach2, etc. These genes play important roles in pathways such as inflammation regulation, immune response, and antigen presentation. Among them, genetic variations in Oas1a and Oas1i are associated with an increased risk of AD, and the Twist1, Dach2, Mnda, and Mefv genes play roles in neurodegenerative diseases or neuroinflammation.

[0087] (4) The KEGG enrichment scatter plot of differentially expressed genes is as shown in Figure 11As shown. The results showed that the pathways mainly enriched by differentially expressed genes between the model group and the treatment group were: antigen processing and presentation, cell adhesion molecules, phagosomes, cellular senescence, herpes simplex virus type 1 infection, complement and coagulation cascades, etc. The above pathways are closely related to the pathogenesis of AD, participate in the pathological progression of AD, and affect neuronal damage by regulating synaptic plasticity, mediating neuroinflammation, maintaining blood-brain barrier function, regulating pro-inflammatory cells, etc.

[0088] In summary, the method for extracting tobacco leaf exosomes of the present invention is simple and easy to operate. After connecting the brain-targeting peptide RVG29 to the surface of the tobacco leaf exosomes for epigenetic modification and then loading donepezil inside, the exosomes can bind to the corresponding specific proteins, making them have active targeting properties and reducing the side effects caused by systemic exposure of the drug. Using microneedles as a drug delivery system, applying the drug-loaded exosomes to the microneedles and adopting the method of nasal administration can enhance the delivery efficiency of the drug through the blood-brain barrier into the central nervous system. While achieving less trauma and reduced side effects, it also makes the therapeutic effect of the drug better. It provides ideas for the treatment of Alzheimer's disease and other brain diseases.

[0089] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, or improvements made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A transnasal microneedle drug delivery system for Alzheimer's disease, characterized in that, It includes a microneedle base and a microneedle tip; the microneedle tip includes a drug-loaded exosome, hyaluronic acid, and polyvinylpyrrolidone; The drug-loaded exosome includes a tobacco leaf exosome, and a brain-targeting peptide RVG29 is connected to the surface of the tobacco leaf exosome, and donepezil is encapsulated inside the tobacco leaf exosome.

2. The transnasal microneedle drug delivery system for Alzheimer's disease according to claim 1, wherein The microneedle base is polyvinylpyrrolidone.

3. The transnasal microneedle drug delivery system for Alzheimer's disease according to claim 1, wherein The tobacco leaf exosome is prepared by the following method: mixing tobacco leaves with water for juicing, then performing gradient centrifugation, and then purifying by sucrose density gradient centrifugation to obtain the tobacco leaf exosome.

4. The preparation method of the transnasal microneedle drug delivery system for Alzheimer's disease according to claim 1, characterized in that, It includes the following steps: (1) Mixing the brain-targeting peptide RVG29 and the tobacco leaf exosome, and performing ultrasonic treatment to obtain a modified exosome; (2) Mixing donepezil with the modified exosome obtained in step (1), incubating, and performing ultrafiltration to obtain a drug-loaded exosome; (3) Mixing hyaluronic acid, polyvinylpyrrolidone, and the drug-loaded exosome obtained in step (2) evenly, adding them into a microneedle mold, and drying and curing to obtain the microneedle tip; (4) Adding polyvinylpyrrolidone to the bottom of the microneedle tip in step (3), and drying and curing to obtain a transnasal microneedle drug delivery system for Alzheimer's disease.

5. The preparation method of the transnasal microneedle drug delivery system for Alzheimer's disease according to claim 4, wherein In step (1), the volume ratio of the brain-targeting peptide RVG29 to tobacco leaf exosomes is 400 - 600 μL: 400 - 600 μL; the concentration of the tobacco leaf exosomes is 6.10×10 11 particles / mL.

6. The preparation method of the transnasal microneedle drug delivery system for Alzheimer's disease according to claim 4, wherein, In step (2), incubate at a temperature of 35 - 40 °C for 3 - 5 h.

7. The preparation method of the transnasal microneedle drug delivery system for Alzheimer's disease according to claim 4, characterized in that, In step (2), use an ultrafiltration tube with Mw = 3 kDa for ultrafiltration.

8. The preparation method of the transnasal microneedle drug delivery system for Alzheimer's disease according to claim 4, wherein, In step (2), the volume ratio of donepezil to the modified exosome is 1 - 2 mL : 400 - 600 μL.

9. The preparation method of the transnasal microneedle drug delivery system for Alzheimer's disease according to claim 4, wherein, In step (3), the mass-to-volume ratio of hyaluronic acid, polyvinylpyrrolidone, and the drug-loaded exosome is 0.1 - 0.3 g : 0.1 g : 0.8 - 1.2 mL.

10. The preparation method of the transnasal microneedle drug delivery system for Alzheimer's disease according to claim 4, wherein In steps (3) and (4), dry and cure at 35 - 40 °C for 20 - 40 min.

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