Use of exosomes loaded with nerve factors on membranes in the preparation of neuroprotective products

Abstract

The application is named application of exosome loaded with nerve factors on membrane in preparation of nerve protection product, and belongs to the technical field of medicine. The technical problem to be solved is that nerve factor delivery technology has serious defects. The technical solution points are that the nerve factors are directly and stably anchored on the surface of the exosome membrane through the transmembrane domain by sorting signal peptide and transmembrane domain, the sorting signal peptide, the transmembrane domain and the N terminal or C terminal of the nerve factors are directly connected or connected through a connecting sequence, and the sorting signal peptide and the transmembrane domain comprise an amino acid sequence as shown in any one of SEQ ID NO. 1-8 or an amino acid sequence with a homology of more than 95% with any one of SEQ ID NO. 1-8.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of exosomes loaded with neurotrophic factors on membranes in the preparation of neuroprotective products. Background Technology

[0002] For understanding the technical content of this invention: Neurotrophin (NT) is a class of protein molecules produced by nerve-innervated tissues (such as muscles) and astrocytes, essential for neuronal growth and survival. Neurotrophin typically enters nerve endings via receptor-mediated endocytosis, then reaches the cell body via retrograde axonal transport, promoting the synthesis of related proteins and thus playing a role in supporting neuronal growth, development, and functional integrity. Although neurotrophin has therapeutic effects in neuroregenerative medicine, it suffers from drawbacks such as inability to cross the blood-brain barrier, low bioavailability when administered systemically, short plasma half-life, poor tissue permeability, and easy dimerization and inactivation at room temperature.

[0003] Recently, exosomes have been used to target the delivery of therapeutic agents to the brain. As a natural nanocarrier, exosomes have a bilayer lipid structure similar to the cell membrane, and have low immunogenicity, high biocompatibility and blood-brain barrier penetration ability, making them an ideal delivery system for neurotherapeutic molecules. The adhesion molecules on their surface help interact with recipient cells. Through genetic engineering, specific targeting ligands can be expressed on the surface of exosomes to achieve targeted delivery to specific cells.

[0004] Relevant patent documents retrieved: The document, published in China (CN117919197A) on December 21, 2023, discloses a low-immunogenicity targeted vesicle delivery system, its preparation method, and its application. The document discloses that the vesicle delivery system achieves targeting through artificial chimeric receptors, including scFv domains that specifically bind to tumor antigens, transmembrane domains that can be enriched in exosomes, and intravesicular domains with tracer functions.

[0005] Relevant non-patent literature retrieved: The journal or book title is *The Protein Journal*, and the article title is "Exosomes with EngineeredBrain Derived Neurotrophic Factor on Their Surfaces Can Proliferate Menstrual Blood Derived Mesenchymal Stem Cells: Targeted Delivery for a Protein Drug," Volume 43, Publication Date: October 13, 2024. This article discloses how genetically engineered exosomes, using the LAMP-2b protein to express brain-derived neurotrophic factor (BDNF) on their surface, can promote the proliferation of menstrual blood-derived mesenchymal stem cells (Mens-MSCs) and explores its potential applications in neuroregenerative medicine.

[0006] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects: The modification method primarily relies on the fusion of the LAMP-2b protein and does not involve more precise targeting mechanisms, such as the screening and optimization of signal peptides. Therefore, its targeting ability may be limited, failing to achieve more precise nerve cell targeting. Furthermore, the existing technology only theoretically demonstrates the feasibility of expressing neurotrophic factors on the surface of exosomes in protecting neurons. Summary of the Invention

[0007] The purpose of this invention is to provide: The application of exosomes loaded with neurotrophic factors on a membrane in the preparation of neuroprotective products, and related technologies, to solve technical problems such as serious defects in neurotrophic factor delivery technology, or combinations thereof.

[0008] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0009] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0010] The definition of standard chemical terms can be found in the reference "Experimental Tutorial of Cell and Molecular Biology", People's Medical Publishing House, Sun Yueping and Xu Weige.

[0011] Unless otherwise stated, conventional methods within the scope of the art, such as plasmid construction, virus packaging and transfection, shall be used.

[0012] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0013] The terms “optional / arbitrary” or “optionally / arbitrarily” mean that the event or condition described below may or may not occur, including both the occurrence and non-occurrence of the event or condition. For example, according to the definition below: for example, “the amino acid sequence of the signal peptide and the transmembrane domain is arbitrarily selected from SEQ ID NO.1-8” means “the amino acid sequence can be SEQ ID NO.1, SEQ ID NO.3 or SEQ ID NO.8”.

[0014] The term "exosome" as used in this article refers to nanoscale vesicles secreted by cells, which are widely distributed in body fluids. They can carry biomolecules such as proteins, lipids, mRNA, and miRNA, and participate in various physiological processes such as intercellular communication, immune regulation, and tissue repair.

[0015] The term "brain-derived neurotrophic factor" used in this article refers to the core members of the neurotrophic factor family, which play a decisive role in neuronal survival, synaptic plasticity, and neural circuit remodeling. Widely distributed in the central nervous system, including the cortex, hippocampus, basal ganglia, and spinal cord, it can specifically regulate glutamatergic neurons, GABAergic neurons, and basal forebrain cholinergic neurons, maintaining synaptic plasticity and mediating learning, memory, and cognitive processes.

[0016] The term "nerve growth factor" used in this article refers to a core member of the neurotrophic factor family, which plays a decisive role in the survival, development, and functional maintenance of sympathetic neurons, sensory neurons (especially nociceptors), and basal forebrain cholinergic neurons.

[0017] The term "signal peptide" as used in this article refers to a short peptide sequence located at the N-terminus of a secretory protein or polypeptide, typically consisting of 10-30 amino acid residues. It plays a crucial role in protein synthesis, transport, and localization, guiding proteins from the cytoplasm to specific organelles outside or inside the cell.

[0018] The term "transmembrane domain" as used in this article refers to a specific amino acid sequence region in a protein that can anchor itself in the phospholipid bilayer of the cell membrane. It plays a crucial role in the structure and function of membrane proteins.

[0019] In a first aspect, the present invention provides the application of exosomes loaded with neurotrophic factors on a membrane in neuroprotection, wherein the membrane of the exosomes includes neurotrophic factors, sorting signal peptides and transmembrane domains, and the neurotrophic factors include, but are not limited to, any one or more of BDNF, NGF, NT-3, and NT-4 / 5.

[0020] The neuroprotection includes: nerve repair and nerve regeneration; The nerve regeneration and nerve repair include at least one of increasing nerve cell activity, increasing nerve cell proliferation capacity, and promoting nerve synapse formation.

[0021] The exosomes are used to prevent and / or treat spinal cord injury, stroke, traumatic brain injury, diabetic peripheral neuropathy, chemotherapy-induced neuropathy, postoperative nerve injury, depression, anxiety, post-traumatic stress disorder, schizophrenia, substance addiction, autism spectrum disorder, attention deficit hyperactivity disorder, cerebral palsy, Rett syndrome, sensorineural hearing loss, vestibular dysfunction, glaucoma, optic neuritis, retinitis pigmentosa, alcoholic neuropathy, uremic neuropathy, vitamin B12 deficiency-related subacute combined degeneration of the spinal cord, chronic pain, enteric neurodevelopmental dysplasia, age-related cognitive decline, or radiation-induced brain injury.

[0022] The exosomes are used to prevent and / or treat Alzheimer's disease by improving the neuronal function and cognitive behavior of the subjects.

[0023] The signal peptide and transmembrane domain include any one of SEQ ID NO.11, SEQ ID NO.12 and SEQ ID NO.8 or include sequences that have more than 90% homology with any one of SEQ ID NO.11, SEQ ID NO.12 and SEQ ID NO.8.

[0024] SEQ ID NO.11: VNDRWKTMGGAAQLEDRPRDKPQRPSCGYVLCTVLLALAVLLAVAVTGAVLFL.

[0025] SEQ ID NO.12: YCLMITFAIFSLIMLVEVAAAIAGYVFRDKVMSEFNNNFRQQMENYPKNNHTASI.

[0026] The sequence including SEQ ID NO.11 includes SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.7.

[0027] The sequences including SEQ ID NO.12 include SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6.

[0028] The connection methods for the technical feature signal peptide, transmembrane domain and neurofactor are selected from: direct connection or connection through a connection sequence.

[0029] The technical feature "direct connection" includes connections to the N-terminus or C-terminus of neural factors.

[0030] Among them, the technical feature "connection sequence" includes, but is not limited to, flexible connection sequence.

[0031] Among them, the technical feature "flexible connection sequence" includes, but is not limited to, (G) x S y ) n x≤4, y≤2, n≤6.

[0032] Among them, technical features "(G x S y ) n "Preferred is GGGSGGGSGGGS (SEQ ID NO.13) or GGGGSGGGGS (SEQ ID NO.14)."

[0033] The preparation of the technically distinctive exosomes includes the following steps: S1: Construct an expression vector, wherein the expression vector comprises a sequence encoding SEQ ID NO.1-8 or having more than 90%, 95%, 98% or 99% homology with any of SEQ ID NO.1-8; S2: Transfect the expression vector into host cells; S3: Isolate and collect exosomes from transfected cells.

[0034] The technical feature expression vector can be a eukaryotic expression vector or a prokaryotic expression vector. The eukaryotic expression vector includes, but is not limited to, any one or more of pFastBac, pEGFP-N1, pcDNA3.1, and pLVX. The prokaryotic expression vector includes, but is not limited to, any one or more of pET28a, pET32a, pGEX-4T, pMAL-p2x, and pMAL-c2X.

[0035] The host cell, as a technical feature, can be a eukaryotic host cell or a prokaryotic host cell. The eukaryotic cell includes, but is not limited to, any one or more of yeast, filamentous fungi, insect cells, mammalian cells, and plant cells. The prokaryotic cell includes, but is not limited to, any one or more of Escherichia coli, Bacillus subtilis, lactic acid bacteria, Streptomyces, Proteus mirabilis, Corynebacterium glutamicum, thermophilic bacteria, cyanobacteria, and halophilic bacteria.

[0036] More specifically, the yeast includes, but is not limited to, any one or more of the following: Pichia pastoris, Saccharomyces cerevisiae, Schizosomalidomiae, Kluyveromyces lactis, or Hansenula polymorpha.

[0037] More specifically, the filamentous fungi include, but are not limited to, any one or more of Trichoderma, Aspergillus niger, or Trichoderma reesei.

[0038] More specifically, the insect cells include, but are not limited to, any one or more of the Sf9, Sf21, or High-5 cell lines.

[0039] More specifically, the mammalian cells include, but are not limited to, any one or more of the following: Chinese hamster ovary cells (CHO), monkey kidney cells (COS), hamster kidney cells (BHK), mouse embryonic fibroblasts (NIH3T3), mouse myeloma cells (SP2 / 0 cells), ExpiCHO-S cell line, or FreeStyle293-F cell line.

[0040] More specifically, the plant cells include, but are not limited to, any one or more of the following: tobacco BY-2 cells or Arabidopsis thaliana cells.

[0041] Among them, the separation methods of exosomes with technical characteristics include, but are not limited to: differential centrifugation, density gradient centrifugation, ultrafiltration, polymer precipitation, immunoaffinity capture, size exclusion chromatography, or microfluidic chip technology.

[0042] Among them, products with technical features include, but are not limited to: vaccines and pharmaceutical compositions.

[0043] The pharmaceutical composition also includes pharmaceutically acceptable excipients.

[0044] The pharmaceutically acceptable excipients include any one or more of the following: binders, fillers, disintegrants, lubricants, preservatives, antioxidants, flavoring agents, fragrances, solubilizers, emulsifiers, solubilizers, or osmotic pressure regulators.

[0045] The excipients include, but are not limited to: lactose, mannitol, methylcellulose, benzoic acid, sorbic acid, food coloring and / or talc.

[0046] The dosage form of the composition includes: liquid dosage form, gas dosage form, solid dosage form or semi-solid dosage form.

[0047] The liquid dosage forms include: solvent-based, aromatic aqueous solutions, tinctures, elixirs, colloidal solutions, pastes, suspensions, or emulsifiers.

[0048] The gaseous dosage form includes: aerosol or spray.

[0049] The solid dosage forms include: powders, pills, tablets, or films.

[0050] The semi-solid dosage forms include ointments, suppositories, or pastes.

[0051] The administration methods of the pharmaceutical composition include oral, injection, implantation, external application, spray, inhalation, or combinations thereof.

[0052] The injection administration methods include, but are not limited to: intraocular injection, intravenous injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, intrathecal injection, intra-articular injection, or intratumoral injection.

[0053] The external application methods include, but are not limited to: mucosal administration or transdermal / local administration.

[0054] The mucosal administration includes, but is not limited to: nasal administration, oral buccal / sublingual administration, and ocular administration.

[0055] The transdermal / local drug delivery includes, but is not limited to, transdermal patch drug delivery.

[0056] The inhalation administration method includes, but is not limited to, nebulized inhalation or metered-dose inhaler inhalation.

[0057] Based on further solutions to the technical problems of the present invention, or simultaneous solutions to multiple technical problems, the preferred solution in the technical solution provided in the first aspect of the present invention includes: The signal peptide and transmembrane domain of any one of SEQ ID NO.1-8 are directly linked to the C-terminus / N-terminus of the neurofactor. This technology not only solves the problem of low delivery efficiency of the neurofactor but also addresses the technical issue of insufficient targeting of the neurofactor. It achieves precise anchoring through highly specific sequences, thereby improving the neurorepair effect.

[0058] The second preferred approach involves using the sorting signal peptide and transmembrane domain shown in SEQ ID NO. 1-8, linked to the N-terminus of BDNF via a flexible linker sequence (SEQ ID NO. 13: GGGSGGGSGGGS or SEQ ID NO. 14:). This approach addresses the insufficient targeting of neurotrophic factors and further solves the problem of poor exosome membrane loading stability. The flexible linker enhances protein conformational flexibility and prolongs the duration of action.

[0059] Examples 1-16 and detection examples 1-2 in this invention at least support the protection scope of claims 1-3.

[0060] Regarding the claim 1: Neuroprotection includes neural repair and neural regeneration: this is summarized from the corresponding technical characteristics (increased cell proliferation rate, increased number of synapses, and increased proportion of preferred social time) in the cell experiments (PC12 cell proliferation and synapse formation) and animal experiments (improved social skills in APP / PS1 mice) of Examples 1-16, through the common feature "recovery of neural function". Those skilled in the art can infer that other indicators reflecting neural repair or regeneration (such as neuronal survival rate and axonal growth length) also fall within the scope of protection.

[0061] The components and sequences on the exosome membrane are summarized from the sequences and linkages of SEQ ID NO. 1-8 used in Examples 1-16, based on the common feature of "sorting signal peptide + transmembrane domain + membrane anchoring system of neurotrophic factor". Sequences with homology exceeding 90%, 95%, 98%, or 99% (such as truncated or conserved amino acid substitution variants of SEQ ID NO. 1-8) are also within the scope of protection of this invention due to their functional equivalence.

[0062] Regarding claim 2: Neurotrophic factors include BDNF, NGF, NT-3, and NT-4 / 5. Examples 1-8, through experiments using BDNF-loaded exosomes (cell proliferation increased by 112%-162.9%), and Examples 9-16, through experiments using NGF-loaded exosomes (cell proliferation increased by 117.1%-136.6%), directly verified the effectiveness of BDNF and NGF. Based on the homology of the neurotrophic factor family, the equivalent effects of NT-3 and NT-4 / 5 can be derived using conventional techniques and are also within the scope of this invention.

[0063] Claim 3 defines the connection method (direct connection or connection sequence) between the sorting signal peptide, transmembrane domain, and neurotrophic factor. Examples 1, 3, 7, 8, 9, 11, 15, and 16 use direct connection, while Examples 2, 4, 5, 6, 10, 12, 13, and 14 use flexible connection sequences (such as GGGSGGGSGGGS). Both methods showed significant neuroprotective effects (e.g., cell proliferation was 162.9% in the direct connection group of Example 1 and 141.5% in the connection sequence group of Example 4), verifying the feasibility of the connection method and supporting the claim.

[0064] The beneficial effects of this invention are as follows: This invention achieves direct and stable anchoring of neurotrophic factors on exosome membranes by screening and optimizing sorting signal peptides and transmembrane domains (SEQ ID NO. 1-8), thereby improving targeting. Experiments show that the proportion of APP / PS1 mice in Example 1 who preferred social interaction time reached 82%, significantly higher than the positive control drug (60.67%).

[0065] Improved stability and bioavailability of neurotrophic factors: This solved the problems of easy inactivation of neurotrophic factors and poor blood-brain barrier penetration. In cell experiments, the proliferation rate of PC12 cells in Example 1 reached 162.9±11.63, which was much higher than that of the control group (100±3.308).

[0066] Extensive neuroprotective applications: It can be used for more than 30 diseases, including spinal cord injury, stroke, and depression. In animal experiments, groups 1 and 4 of Examples showed better improvement in social abilities in Alzheimer's disease model mice than the positive control drug, demonstrating its potential in neurodegenerative diseases. Attached Figure Description

[0067] Figure 1 The proliferation rate of PC12 cells was detected using CCK8 in Example 1.

[0068] Figure 2 To detect synapse formation in PC12 cells as shown by immunofluorescence in Example 1.

[0069] Figure 3 This study aims to statistically analyze the proportion of social-preferred time in each group of the three boxes in Case 2. Detailed Implementation

[0070] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0071] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0072] Basic Implementation Methods for preparing engineered exosomes: (1) Construction of expression vector: including the exosome sorting signal peptide encoding gene, the transmembrane sequence encoding gene and the full-length neurotrophic factor encoding gene. The exosome sorting signal peptide encoding gene and the transmembrane sequence encoding gene were synthesized by a gene synthesis company. Homologous recombination cloning was used to clone the exosome sorting signal peptide encoding gene, the transmembrane sequence encoding gene and the recombinant BDNF protein expression sequence into the vector pcDNA3.1(+) plasmid to form the basic vector of the genetically engineered exosome expression plasmid.

[0073] (2) Plasmid transformation and extraction: Following standard plasmid transformation procedures, the constructed recombinant plasmid was transformed into DH5α Escherichia coli competent cells. The next day, a single colony was picked and inoculated into 3 mL of LB medium, and amplified at 37°C and 200 rpm. After 12 hours, the bacterial culture was inoculated into 200 mL of LB medium and amplified a second time at 37°C and 200 rpm. After 12 hours, plasmid was extracted using a plasmid DNA ultra-rapid high-volume extraction kit (Jianshi Bio, TD423) according to the instructions. The concentration of the extracted plasmid was measured, and the sample was filtered through a 0.22 μm filter for sterilization.

[0074] (3) Detection of target protein: Transfect the correctly sequenced vector into 293T cells. Mix the plasmid with the transfection reagent (Sinofection Transfection Reagent, sinobiological, STF02) according to the instructions and add it into 293T cells. After 48 hours of transfection, WB is used to detect whether the corresponding neurotrophic factor overexpression band appears. If the expression is successful, it can be used for the next experiment.

[0075] (4) Exosome isolation and purification Cell supernatant was collected and subjected to gradient centrifugation at 300×g for 10 min, 2000×g for 20 min, and 10000×g for 60 min to remove cell debris from the cell culture medium. The cell supernatant was concentrated using a hollow fiber membrane column (Cobati, Pilot+, PES, 100KD) and then purified using a gel chromatography column (Focurose 4FF, highly cross-linked 4% agarose, particle size range 45-165µm). The gel column specifications were 16*700, column volume was approximately 100mL, sample loading volume was 1 / 5-1 / 20, sample loading flow rate was 1mL / min, elution buffer was 20mM PBS buffer, elution flow rate was 2mL / min, and the UV detector wavelength was set to 260-280nm to collect the elution peaks. The purified exosomes were named target exosomes.

[0076] Examples 1-8 This application provides various sorting signal peptides and transmembrane domains for enriching neurotrophic factors on exosome membranes. The sorting signal peptide, transmembrane domain, and recombinant BDNF sequence are directly linked at the N-terminus or C-terminus, or linked through a linker sequence. The amino acid sequences of the sorting signal peptide and transmembrane domain are shown in Table 1. Table 1

[0077] BDNF (SEQ ID NO.9): MTILFLTMVISYFGCMKAAPMKEANIRGQGGLAYPGVRTHGTLESVNGPKAGSRGLTSLADTFEHVIEELLDEDQKVRPNEENNKDADLYTSRVMLSSQVPLEPPLLFLLEEYKNYLDAANMSM RVRRHSDPARRGELSVCDSISEWVTAADKKTAVDMSGGTVTVLEKVPVSKGQLKQYFYETKCNPMGYTKEGCRGIDKRHWNSQCRTTQSYVRALTMDSKKRIGWRFIRIDTSCVCTLTIKRGR.

[0078] Table 2 Examples 1-8

[0079] The preparation of exosomes loaded with BDNF on the membrane using different signal peptides and transmembrane domains is described in the basic embodiments.

[0080] Examples 9-16 In Examples 9-16, the neurotrophic factor used was NGF, whose amino acid sequences are as follows: NGF (SEQ ID NO.10): MSMLFYTLITAFLIGIQAEPHSESNVPAGHTIPQAHWTKLQHSLDTALRRARSAPAAAIAARVAGQTRNITVDPRLFKKRRLRSPRVLFSTQPPREAADTQDLDFEVGGAAPFNRTHRSKR SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA.

[0081] Table 3 Examples 9-16

[0082] According to the basic embodiments, exosomes loaded with NGF on the membrane were prepared by using different signal peptides and transmembrane domains.

[0083] Comparative Example 1 The difference from Example 1 is that the expression vector does not contain the sorting signal peptide coding gene and the transmembrane sequence coding gene, but only carries the full-length coding gene for the neurotrophic factor BDNF. Exosomes are prepared according to the steps described in the basic example to construct an exosome containing the neurotrophic factor BDNF.

[0084] Example 1: Cell Experiment Table 4 Grouping conditions

[0085] 1. Assay for cell viability and proliferation capacity (CCK-8 assay kit) PC12 cells were used at a rate of 1×10 5 Cells were seeded at a density of 100 μL / mL in 96-well plates, with 100 μL of cell suspension added to each well. After cell adhesion, cells were treated according to the grouping conditions for 96 hours. Subsequently, 10 μL of LCK-8 solution was added to each well, and the cells were incubated at 37°C for 3 hours. The absorbance (OD value) at 450 nm was measured using a microplate reader, and cell proliferation activity was calculated from the OD value. This method was used to evaluate the viability and proliferation capacity of cells under different treatment conditions. The experimental results are shown in [Figure number missing]. Figure 1 See Table 5.

[0086] Table 5 Results of cell proliferation experiment

[0087] 2. Synapse formation PC12 cells in good growth condition were divided into groups of 1×105 Seeds were inoculated into sterile cell slides at a density of cells / well, and cultured in complete culture medium at 5% CO2 and 37°C for 24 hours. EV samples were added at a concentration of 50 μg / ml, and cultured for another 72 hours. The supernatant was removed, and the cells were washed three times with PBS. After fixation with 4% paraformaldehyde for 15 minutes, the paraformaldehyde was removed, and the cells were permeabilized with 1% Triton X-100 for 10 minutes. The cells were washed three times with PBS, blocked with 5% goat serum for 60 minutes, washed three times with PBS, and incubated overnight at 4°C with β3-Tubulin (D65A4) XP® Rabbit mAb (CST, 5666T) 1:100. The cells were washed three times with PBS, and then incubated with fluorescent secondary antibody (donkey). Anti-rabbit488(GFP)IgG1 (Jackson, 711-545-152) (1:200) was incubated for 2 h, washed three times with PBS, incubated with DAPI dye for 10 min, washed three times with PBS, mounted, and the state of synapse formation was observed under a two-photon laser confocal scanning microscope (ZEISS). This method was used to evaluate the growth of neurites under different treatment conditions. The experimental results are shown in [Figure number missing]. Figure 2 .

[0088] According to the cell experiment results of each embodiment, Examples 1-16 all had a certain positive effect on cell proliferation and neural synapse formation. Among them, Examples 1 and 4 had significant effects on promoting cell proliferation and synapse formation. These two groups were selected for subsequent animal experiments.

[0089] Example 2: Mouse Experiment Mouse model: APP / PS1 mouse model, purchased from Spifor (Beijing) Biotechnology Co., Ltd.

[0090] After a one-week acclimatization period, the mice were kept under conditions of 25±1℃, 55% humidity, and natural darkness / light. After one week of acclimatization, they were randomly divided into 4 groups of 5 mice each and treated according to the treatments described in Table 6.

[0091] Table 6. Treatment conditions for the experimental group

[0092] The processing period is 28 days. After 28 days, a three-box social experiment will be conducted, with the following specific conditions: 1. Experimental Equipment: The experimental setup consists of three rectangular boxes, each measuring 19×45cm. The partitions between the boxes are made of transparent resin glass, with a passageway connecting the three boxes. A uniform metal cage, large enough to hold one mouse, is placed in the center of each of the left and right boxes.

[0093] 2. Pre-test adaptation: Place the mice in the behavioral testing room for half an hour to adapt.

[0094] 3. Test Phase 1: Separate the three boxes with transparent glass resin plates, and place the test mouse (Subjectmice) in the middle box to adapt for 5 minutes; randomly place Stranger1 into the metal cage in the left or right box, leaving the metal cage in the other box empty; remove the glass resin plates separating the boxes, allowing the test mouse to move freely in the three boxes for 10 minutes; immediately begin taking pictures and recording relevant parameters.

[0095] Parameter 1: The duration of direct contact between the Subject mice and the Stranger1 or an empty metal cage, with the contact area defined as 3–5 cm around the metal cage.

[0096] Parameter 2: The duration for which a mouse is in each box. A mouse is considered to be in a box when its head and four paws are in that box.

[0097] Test Phase 2: A second unfamiliar mouse of the same species (Stranger2) was placed in an empty metal cage in the first phase of the experiment. The time of contact between the Subject mice and Stranger1 and Stranger2 was recorded for ten minutes.

[0098] Phase 3 of the test: 24 hours after the completion of Phases 1 and 2, the Stranger 2 mice were replaced with another new, unfamiliar Stranger 3 mouse. The contact time between the test mouse and Stranger 1 and Stranger 3 was recorded for 5 minutes. If the test mouse showed no social curiosity towards Stranger 3 and the difference in contact time between the two mice was not significant, then its social memory ability was weak. The experimental results are shown below. Figure 3 See Table 7.

[0099] Table 7 Evaluation of Experimental Results: Percentage of Preferred Social Time for Three-Box Social Interactions (%)

[0100] according to Figure 3 It can be seen that the positive control group, Example 1 group, and Example 4 group all significantly improved the symptoms of Alzheimer's disease in mice, and the exosomes in Example 1 and Example 4 were more effective than the positive control drug. This indicates that the exosomes with surface-loaded neurotrophic factors provided by the present invention have excellent technical effects in nerve repair and nerve regeneration.

[0101] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. The use of membrane-loaded exosomes carrying neurotrophic factors in the preparation of pharmaceutical compositions for treating Alzheimer's disease, characterized in that, The membrane of the exosome includes: a neurotrophic factor, a sorting signal peptide, and a transmembrane domain, wherein the neurotrophic factor is BDNF, and the sequence of the signal peptide and the transmembrane domain is any one of SEQ ID NO: 1-8; The sorting signal peptide, transmembrane domain, and neurotrophic factor are directly connected at the N-terminus or C-terminus, or through a linking sequence. The connection sequence includes flexible connection sequences (G). x S y ) n x≤4, y≤2, n≤6; The preparation of the exosomes includes the following steps: S1: Construct an expression vector, the expression vector containing sequences encoding SEQ ID NO.1-8; S2: Transfect the expression vector into host cells; S3: Isolate and collect exosomes from transfected cells.

2. The application according to claim 1, characterized in that, In S1, the expression vector is a eukaryotic expression vector or a prokaryotic expression vector. The eukaryotic expression vector is selected from any one or more of pFastBac, pEGFP-N1, pcDNA3.1, and pLVX. The prokaryotic expression vector is selected from any one or more of pET28a, pET32a, pGEX-4T, pMAL-p2x, and pMAL-c2X.

3. The application according to claim 1, characterized in that, In S2, the host cell is a eukaryotic host cell or a prokaryotic host cell. The eukaryotic cell is selected from any one or more of yeast, filamentous fungi, insect cells, mammalian cells, and plant cells. The prokaryotic cell is selected from any one or more of Escherichia coli, Bacillus subtilis, lactic acid bacteria, Streptomyces, Proteus mirabilis, Corynebacterium glutamicum, thermophilic bacteria, cyanobacteria, and halophilic bacteria.

4. The application according to claim 1, characterized in that, In S3, the methods for separating exosomes include: differential centrifugation, density gradient centrifugation, ultrafiltration, polymer precipitation, immunoaffinity capture, size exclusion chromatography, or microfluidic chip technology.

5. The application according to claim 1, characterized in that, The pharmaceutical composition also includes pharmaceutically acceptable excipients.

6. The application according to claim 5, characterized in that, The pharmaceutically acceptable excipients include any one or more of the following: binders, fillers, disintegrants, lubricants, preservatives, antioxidants, flavoring agents, fragrances, solubilizers, emulsifiers, solubilizers, or osmotic pressure regulators.

7. The application according to claim 1, characterized in that, The dosage form of the pharmaceutical composition includes: liquid dosage form, gas dosage form, solid dosage form, or semi-solid dosage form.

8. The application according to claim 7, characterized in that, The liquid dosage forms include: solvent-based, aromatic aqueous solutions, tinctures, elixirs, colloidal solutions, pastes, suspensions, or emulsifiers.

9. The application according to claim 1, characterized in that, The pharmaceutical composition may be administered orally, by injection, implantation, external application, spray, inhalation, or a combination thereof.

10. The application according to claim 9, characterized in that, The injections include: intraocular injection, intravenous injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, intrathecal injection, intra-articular injection, or intratumoral injection.

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