Engineered umbilical cord stem cell exosomes carrying siCCR5, their preparation method, and their application in the treatment of Alzheimer's disease.

Abstract

This invention provides an engineered umbilical cord stem cell exosome carrying siCCR5, its preparation method, and its application in the treatment of Alzheimer's disease, belonging to the field of biomedical technology. This invention provides a method for preparing a new drug for treating Alzheimer's disease. First, a lipid membrane is prepared, then the lipid membrane is dissolved, and siCCR5 and umbilical cord mesenchymal stem cell exosomes are added. The siCCR5 is then delivered into the umbilical cord mesenchymal stem cell exosomes using a cationic liposome extrusion method, resulting in engineered umbilical cord stem cell exosomes carrying siCCR5. The engineered exosomes prepared by this invention can repair brain tissue through tissue regeneration and regulate the brain microenvironment without producing toxic side effects on the body. Furthermore, the engineered exosomes carrying siCCR5, which targets specific genes, have stronger targeted therapeutic efficacy than ordinary hUCMSC-EVs, and can more effectively exert anti-inflammatory effects, improving the progression of Alzheimer's disease.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an engineered umbilical cord stem cell exosome carrying siCCR5, its preparation method, and its application in the treatment of Alzheimer's disease. Background Technology

[0002] Alzheimer's disease is a neurodegenerative disease associated with neuronal damage and is a leading cause of dementia. The incidence of Alzheimer's increases with age due to the imbalance of protein homeostasis caused by aging. In 2019, Alzheimer's disease was listed as the sixth leading cause of death in the United States. Furthermore, COVID-19 has also significantly impacted Alzheimer's mortality rates. It is estimated that by 2060, 13.8 million people in the United States will have Alzheimer's disease, placing a huge healthcare burden on families and society and hindering societal progress.

[0003] Pathologically, Alzheimer's disease is characterized by amyloid plaques (Aβ) and neurofibrillary tangles (NFT) formed by phosphorylated tau protein; furthermore, soluble amyloid β oligomers (AβO) can cause neuronal dysfunction, activate glial cells in the brain, and trigger chronic neuroinflammation.

[0004] The U.S. Food and Drug Administration (FDA) has approved several Alzheimer's disease treatments, including memantine, donepezil, galantamine, and rivastigmine. These drugs are cholinesterase inhibitors, which alleviate cognitive impairment by inhibiting the breakdown of acetylcholinesterase. However, due to the complex pathogenesis of Alzheimer's disease, there are currently no effective treatments or drugs worldwide that can fundamentally cure it; they can only alleviate related symptoms. Therefore, new treatments for Alzheimer's disease are urgently needed.

[0005] In recent years, mesenchymal stem cells (MSCs) have been considered a novel therapy for Alzheimer's disease due to their lack of immune rejection, anti-inflammatory properties, and wide differentiation potential, and have been used in clinical trials. MSCs primarily function in neurodegenerative diseases through three mechanisms: secreting growth factors for immune regulation, suppressing neuroinflammation, and paracrine release of exosomes. Studies have shown that MSCs mainly exert their inhibitory effect on Alzheimer's disease through paracrine release of exosomes, rather than through the cells themselves. Exosomes contain proteins, lipids, DNA, mRNA, and miRNA, playing a crucial role in cell communication. They do not produce toxic side effects on the body and can repair brain tissue through tissue regeneration, regulate the brain microenvironment, and suppress inflammation. Therefore, exosomes show great promise in the clinical application of Alzheimer's disease treatment.

[0006] Chemokine CC subfamily receptor 5 (CCR5), a cell membrane protein belonging to the G protein-coupled receptor family, is an important receptor for leukocyte activation and mobilization, including in monocyte lineage cells. In the central nervous system, microglia show high expression levels of CCR5. CCR5 activation triggers various signaling cascades that alter cellular calcium flux and chemotaxis, affecting cellular metabolic activities such as proliferation and apoptosis, promoting the migration of activated cells, inducing the release of pro-inflammatory factors, and stimulating downstream immune cells. Recent research published in Cell indicates that CCR5 plays a crucial role in the regulation of learning and memory. Delayed expression of CCR5 in the CA1 region of mice leads to decreased neuronal excitability, closes the time window for memory connections, and causes memory impairment in aged mice. However, the binding of CCR5 to exosomes has not yet been used to treat Alzheimer's disease.

[0007] Therefore, how to modify exosomes to serve as natural carriers for siCCR5, and construct engineered exosomes capable of gene editing to achieve cell and gene therapy, is crucial for the treatment of Alzheimer's disease. Summary of the Invention

[0008] The purpose of this invention is to provide an engineered umbilical cord stem cell exosome carrying siCCR5, its preparation method, and its application in the treatment of Alzheimer's disease. It can be used to treat Alzheimer's disease, realize cell and gene therapy, significantly improve patients' memory ability, and enhance spatial memory ability.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0010] This invention provides a method for preparing engineered exosomes, comprising the following steps:

[0011] (1) Preparation of liposome membrane: DLin-MC3-DMA was dissolved in an organic solvent (chloroform:methanol = 9:1), cholesterol and DOPC were further dissolved to prepare a liposome solution, the organic solvent was removed by rotary evaporation, and the solution was dried under an inert gas environment to obtain a liposome membrane;

[0012] (2) Preparation of engineered exosomes: The lipid membrane was hydrated with phosphate buffer, and after sonication, siCCR5 and umbilical cord mesenchymal stem cell exosomes were added to obtain a mixed solution. The mixed solution was hydrated, and under heat preservation conditions, it was filtered, squeezed and dialyzed to obtain engineered exosomes.

[0013] Furthermore, in step (1), the organic solvent is a mixed solvent of chloroform and methanol prepared in a volume ratio of 9:1; the molar ratio of DLin-MC3-DMA: cholesterol: DOPC is 25-34.5:35-65:1.5-40.

[0014] Furthermore, in step (2), the mass-to-volume ratio of the lipid membrane to PBS phosphate buffer during hydration and dissolution is 100:3; the mass ratio of siCCR5: umbilical cord mesenchymal stem cell exosomes: lipid membrane is 2:1:50.

[0015] Furthermore, in step (2), the temperature of the heat preservation is 40-50°C.

[0016] Furthermore, in step (2), the siCCR5 can target CCR5 for knockdown, and the siCCR5 is composed of the sense sequence shown in SEQ ID NO.1 and the antisense sequence shown in SEQ ID NO.2.

[0017] Furthermore, in step (2), the extrusion step is as follows:

[0018] The mixed solution was first squeezed through a filter with a pore size of 1.0 μm, then squeezed a second time through a filter with a pore size of 0.1 μm, and finally squeezed a third time through a filter with a pore size of 0.05 μm.

[0019] Furthermore, the filters used in the three extrusions are polycarbonate filters, and the number of extrusions is 2 to 5 times.

[0020] Furthermore, in step (2), the dialysis procedure is as follows:

[0021] Dialysis was performed overnight at 4°C using a dialysis cartridge with a pore size of 100 kDa, and the pH was adjusted to 7.2–7.4.

[0022] The present invention also provides a method for preparing engineered exosomes.

[0023] This invention also provides the application of engineered exosomes in the preparation of drugs for treating Alzheimer's disease.

[0024] The engineered exosomes described in this invention have the following advantages compared to existing technologies:

[0025] This invention utilizes lipids to modify exosomes, employing a cationic liposome extrusion method to deliver siCCR5 into the exosomes, resulting in engineered umbilical cord stem cell exosomes carrying siCCR5. The engineered exosomes prepared by this invention do not produce toxic side effects on the body, can repair brain tissue through tissue regeneration, regulate the brain microenvironment, inhibit inflammation, and, carrying siCCR5 targeting specific genes, exhibit stronger targeted therapeutic efficacy and more efficient anti-inflammatory effects than ordinary hUCMSC-Exos, improving the progression of Alzheimer's disease, achieving cell and gene therapy, significantly improving patients' memory abilities, and enhancing spatial memory. Attached Figure Description

[0026] 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.

[0027] Figure 1 This is a diagram showing the results of flow cytometry identification of cell surface marker proteins in Example 1;

[0028] Figure 2 This is a morphological image of MSC-3P1 cells cultured on day 2 in Example 1.

[0029] Figure 3 The results of flow cytometry identification of surface markers of umbilical cord mesenchymal stem cells in Example 1;

[0030] Figure 4 The staining results for induction of osteogenic and adipogenic differentiation of umbilical cord mesenchymal stem cells in Example 1;

[0031] Figure 5 This is a flowchart of the ultracentrifugation separation of exosomes in Example 1;

[0032] Figure 6 Electron micrograph of hUCMSCs-Exos in Example 1;

[0033] Figure 7 The particle size distribution of hUCMSCs-Exos in Example 1 is shown.

[0034] Figure 8 The expression of hUCMSCs-Exos marker protein in Example 1;

[0035] Figure 9 miRNA sequencing analysis in hUCMSCs-Exos in Example 1

[0036] Figure 10This refers to the engineered exosome technology route in Example 1;

[0037] Figure 11 This is a flowchart of the extrusion-engineered exosome process in Example 1;

[0038] Figure 12 Electron micrograph of the engineered exosomes in Example 1;

[0039] Figure 13 Particle size analysis of engineered exosomes in Example 1;

[0040] Figure 14 To illustrate how hUCMSCs-Exos reduced CCR5 expression in microglia in Experiment 1, we show: a) differential gene enrichment map; b) gene expression heatmap; c) GO enrichment analysis pathway map; and d) changes in CCR5 expression at the protein and gene levels in BV2 cells after treatment with hUCMSCs-Exos.

[0041] Figure 15 For the identification of the transgenic mice in Experiment Example 1;

[0042] Figure 16 The results of the new object recognition experiment and the new location recognition experiment of APP / PS1 mice in Experiment Example 1 are shown in the figure;

[0043] Figure 17 The image shows the results of the Morris water maze for APP / PS1 mice in Experiment 1. In the image, a represents the escape delay time of the mice, b represents the movement trajectory of the mice, c represents the number of times the mice crossed the maze, d represents the time the mice spent in the target quadrant, e represents the total swimming distance of each group of mice, and f represents the swimming speed of each group of mice. Detailed Implementation

[0044] 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.

[0045] 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. Every smaller range between any stated value or intermediate value within a stated range, and 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] This invention prepares an engineered exosome, specifically comprising the following steps;

[0050] (1) Preparation of liposome membrane: Lipids are dissolved in an organic solvent, and cholesterol and DOPC are further dissolved to prepare a liposome solution; the organic solvent is removed by rotary evaporation, and the solution is dried under an inert gas environment to obtain a lipid membrane;

[0051] (2) Preparation of engineered exosomes: The lipid membrane was hydrated with phosphate buffer, and after sonication, siCCR5 and umbilical cord mesenchymal stem cell exosomes were added to obtain a mixed solution. The mixed solution was hydrated, and under heat preservation conditions, it was filtered, squeezed and dialyzed to obtain engineered exosomes.

[0052] In this invention, siCCR5 is a short nucleic acid chain designed to target and knock down CCR5, comprising:

[0053] sense(SEQ ID NO.1):

[0054] GGUCAGUUCCGACCUAUAGTT;

[0055] antisense (SEQ ID NO.2):

[0056] CUAUAGGUCGGAACUGACCTT.

[0057] Example 1

[0058] Embodiment 1 of the present invention provides a method for preparing engineered exosomes, which specifically includes the following steps:

[0059] (1) Isolation and culture of umbilical cord mesenchymal stem cells

[0060] A. Isolation of primary umbilical cord mesenchymal stem cells:

[0061] Cut fresh, healthy umbilical cord tissue into 2-3 cm segments, remove the Wharton's jelly portion, and then finely chop the Wharton's jelly. Transfer the jelly to a culture flask, add an appropriate amount of culture medium, and place it in a CO2 incubator. Do not shake the culture flask during this time to avoid tissue fragment detachment. When the cells that have emerged cover approximately 90% of the bottom area of ​​the cell culture flask, passage the primary cells.

[0062] B. Passaging of umbilical cord mesenchymal stem cells

[0063] When the cells reach 90% confluence, discard the old culture medium in the culture flask and wash the cells with an appropriate amount of PBS. Aspirate the PBS, add an appropriate amount of stem cell-mild trypsin, and digest until the cells detach from the bottom of the culture flask. Stop digestion by adding an equal volume of serum-free complete culture medium. Use a pipette to agitate the bottom of the flask and transfer the cell suspension to a centrifuge tube. Centrifuge at 1000 rpm for 5 minutes at room temperature. Discard the supernatant, resuspend the cells in an appropriate amount of complete culture medium, and count the cells. Add an appropriate amount of complete culture medium to a clean culture flask, seed the cells, mix thoroughly, and incubate at 37°C in a CO2 incubator to complete cell passage.

[0064] C. Identification of umbilical cord mesenchymal stem cells

[0065] 1) Morphological identification

[0066] Cell morphology was observed under a microscope, and the results were as follows: Figure 2 As shown.

[0067] Figure 2 The cells showed fibroblast-like growth.

[0068] 2) Marker identification

[0069] The expression levels of cell surface markers CD44, CD73, CD90, CD105, CD34, and CD45 were measured by flow cytometry. An IgG isotype control was used as a negative control. The results are as follows: Figure 1 As shown.

[0070] Figure 3 The results showed that the expression rates of positive markers for mesenchymal stem cells, CD44, CD73, CD90, and CD105, were all >95%, while the expression rates of negative markers for mesenchymal stem cells, CD34 and CD45, were less than 1.0%. The identification results met the standards, indicating that the cells were mesenchymal stem cells.

[0071] 3) Identification of osteogenic and adipogenic differentiation of stem cells

[0072] MSCs possess differentiation potential, meaning they can differentiate into osteoblasts and adipocytes under specific induction conditions. Their differentiation capacity after in vitro adipogenic and osteogenic induction can be assessed using specific staining.

[0073] Steps for osteogenic induction, differentiation, and identification:

[0074] Mesenchymal stem cell inoculation: Cells in the logarithmic growth phase are harvested and inoculated at a rate of 2 × 10⁻⁶ cells / year. 4 cells / cm 2 Cells were seeded at a density suitable for osteogenic differentiation into six-well plates and cultured at 37°C with 5% CO2 until confluence reached 60-70%. The supernatant was discarded, and induction differentiation medium was added. The osteogenic induction medium was DMEM complete medium (DMEM + 10% FBS + 1% P / S + 200 μM sodium ascorbate + 100 μM β-glycerophosphate + 0.1 μM dexamethasone), and the adipogenic differentiation medium was (DMEM + 10% FBS + 1% P / S + 1 μM dexamethasone + 0.5 mM 3-isobutyl-1-methylxanthine + 200 μM indomethacin + 10 μg / mL insulin). Cell differentiation induction: The osteogenic / adipogenic induction medium was changed every 2-3 days, and the cells were cultured at 37°C with 5% CO2 for approximately 14-21 days, observing cell morphological changes. The induction was terminated based on cell condition, and staining identification was performed. Cell fixation: Aspirate the culture medium and wash once with an appropriate amount of 1×PBS. Discard the fixative and cover the bottom of the culture dish with 2mL of 4% neutral formaldehyde solution. Fix at room temperature for 30min. Discard the fixative and wash twice with PBS.

[0075] Osteogenic differentiation Alizarin Red staining: Before use, allow the cells to stand at room temperature. Add 1 mL of 1% Alizarin Red staining solution (pH 4.2) to each well and stain at 37°C for 30 min. Aspirate the Alizarin Red staining solution, wash twice with PBS, and add an appropriate amount of PBS to prevent cell drying. Adipogenic differentiation Oil Red O staining: Prepare Oil Red O working solution using ddH2O and Oil Red O stock solution (Oil Red O stock solution:ddH2O = 3:2). After preparation, filter through a filter membrane and prepare fresh before use. Add 1 mL of working solution to the cleaned induction wells, incubate at 37°C for 30 min, aspirate the Oil Red O working solution, wash twice with PBS, and add an appropriate amount of PBS to prevent cell drying.

[0076] Induction Assessment: The staining effects of osteogenic and adipogenic differentiation were observed under a microscope, and images were acquired and the induction process was assessed. When induction was successful, calcified nodules would bind to Alizarin Red dye and appear red or orange-red; osteogenic nodules formed after differentiation would be stained purple; and adipogenic lipid droplets would be stained red. Figure 4 As shown.

[0077] (2) Isolation of umbilical cord mesenchymal stem cell exosomes (hUCMSCs-EVs)

[0078] A. Expand the culture of umbilical cord mesenchymal stem cells from step (1), according to... Figure 5 The centrifugation steps shown are as follows: the passaged cells are centrifuged at 300g for 5 min, the cells are removed, and then centrifuged at 2000g for 20 min. The supernatant is collected and filtered through a 0.22μm filter membrane. The supernatant is then dissolved in PBS and centrifuged at 120,000g for 120 min to obtain exosomes hUCMSCs-EVs.

[0079] B. Exosome identification

[0080] 1) Electron microscopy observation

[0081] The isolated exosomes were observed using electron microscopy, and the results are as follows: Figure 6 As shown.

[0082] Figure 6 The results show that hUCMSCs-EVs exhibit a typical disc-shaped bilayer membrane structure.

[0083] 2) Exosome particle size

[0084] The particle size of the isolated exosomes was detected, and the results are as follows: Figure 7 As shown.

[0085] Figure 7 The results of five replicates of nanoparticle size analysis showed that the diameter of the obtained exosomes was distributed at around 100 nm, which is within the specified particle size range for exosomes.

[0086] 3) Results of Western Blotting Detection of Exosome Marker Proteins

[0087] TSG101, CD63, and ALIX proteins were selected as marker proteins for exosomes. TSG101 is an ESCRT complex-related protein. CD63 is a member of the four-transmembrane protein family, directly involved in the sorting of ESCRT-dependent and ESCRT-independent extracellular vesicle contents. ALIX is directly involved in the process of vesicle cleavage and detachment from the plasma membrane to form independent membrane structures during vesicle formation. Therefore, the expression of these proteins can be used to identify whether an exosome is present. The results are as follows: Figure 8 As shown.

[0088] Figure 8 The results showed that the extracted exosomes successfully expressed three marker proteins.

[0089] 4) High-throughput sequencing results of miRNAs in hUCMSCs-EVs

[0090] miRNAs are key components of exosomes. This invention employs high-throughput sequencing to determine their types and amounts, and the results are as follows: Figure 9 As shown.

[0091] Figure 9 Studies have shown that miR-21 and miR-146, which are present in higher concentrations, are associated with multiple signaling pathways, regulate the expression of downstream target genes, and affect cellular metabolism. Currently, a large number of studies are focusing on miRNAs as targets for research in various metabolic-related diseases.

[0092] (3) Construction of engineered exosomes

[0093] according to Figure 10 , 11 The process shown is used to prepare engineered exosomes. The specific preparation method is as follows:

[0094] A. Preparation of liposome solution: Lipids were dissolved in a chloroform / methanol mixture (9 / 1; v / v) and prepared into a liposome solution with a concentration of 4 nmol and a total volume of 1 mL at a molar ratio of 25:35:40 (DLin-MC3-DMA:DOPC:cholesterol).

[0095] DLin-MC3-DMA was purchased from MCE Biotech Ltd.; DOPC was purchased from Maclean Reagent Ltd.; and cholesterol was purchased from Sima Biotechnology Ltd.

[0096] B. Preparation of liposome membranes using a rotary evaporator: Add the prepared liposome solution to a 5 mL round-bottom flask. Evaporate the organic solvent in situ under vacuum using a rotary evaporator at 45 °C, and then dry the resulting lipid membrane under a nitrogen flow for approximately 20 minutes.

[0097] C. Hydration and compression: Hydrate the lipid membrane with phosphate buffer (reagents and preparation ratios are as follows: ...). Figure 2 (As shown), sonicate for 5 min; add a mixture of siCCR5 (Suzhou Gemma Gene Co., Ltd.) and umbilical cord mesenchymal stem cell exosomes at a mass ratio of 2:1, and then fully hydrate and dissolve in a siCCR5, exosome, and total lipid membrane ratio of 2:1:50. Maintain the suspension at 45℃, then squeeze five times through a 1.0 μm polycarbonate filter, then five times through a 0.1 μm polycarbonate filter, and finally five times through a 0.05 μm polycarbonate filter using a hand extruder. Subsequently, dialyze the liposomes overnight at 4℃ using a 100 kDa dialysis chamber (Slide-A-Lyzer G210K, 22.5-30 μM regenerated cellulose membrane) to change the pH to 7.4 and remove uncoated siRNA, obtaining engineered exosomes.

[0098] (4) Detection of engineered exosomes

[0099] A. Electron microscopy was performed on engineered exosomes, and the results are as follows: Figure 12 As shown.

[0100] Figure 12 The hybrid particles exhibit a near-spherical shape, a typical "cup-shaped" structure, and a size primarily between 30-150 nm, consistent with the morphological identification range of hybrid particles. Electron microscopy images confirm the effective hybridization of siCCR5 in the liposomal exosomes.

[0101] B. Engineered exosome particle size

[0102] Since the hybrid particles are derived from the hybridization of exosomes, siRNA, and lipid particles, and these three types of particles have different sizes, this invention performs nanoparticle size analysis on the hybridized samples to determine their purity. The results are as follows: Figure 13 As shown.

[0103] Figure 13 The engineered exosomes showed a relatively uniform distribution, with particle sizes ranging from 90 to 100 nm, consistent with particle size identification.

[0104] Example 2

[0105] Example 2 of this invention provides a method for preparing engineered exosomes, which differs from Example 1 in that the ratio of exosomes to total lipids in Example 2 is 1:25.

[0106] Experimental Example 1

[0107] Experimental Example 1 of this invention tested the therapeutic effect of engineered exosomes prepared in Example 1 on Alzheimer's disease. The specific method is as follows:

[0108] (1) Establishment and detection of cell models

[0109] Since Alzheimer's disease is closely related to microglia, the deposition of amyloid plaques in the brain triggers an inflammatory response in microglia, releasing various inflammatory factors that migrate to, phagocytose, and clear the plaques, thus inducing an immune response. Therefore, this invention uses LPS to establish an inflammatory model of microglia.

[0110] BV2 microglia were treated at a rate of 1×10 5Cells were passaged and seeded at a density of cells / well (6-well plate) in three groups: control group (normal), LPS inflammation model group, and EVs exosome treatment group. After 12 hours of cell adhesion, the LPS inflammation model group and the EVs exosome group were treated with 1 μg / ml LPS for 24 hours. After 24 hours, the culture medium of the EVs group was replaced with basal medium, and 20 μg / ml hUCMSC-EVs were added for 24 hours. Results are shown in [Figure showing results]. Figure 14 .

[0111] This invention uses qPCR to detect the expression of inflammatory factors. Figure 14 The results showed that treatment with 1 μg / ml LPS for 24 h significantly increased the expression of inflammatory factors. Exosome treatment significantly reduced the expression of inflammatory factors, indicating that exosome treatment inhibited the inflammatory response of microglia. Simultaneously, hUCMSC-EVs treatment promoted the migration ability of microglia, facilitating their movement to amylose deposition sites for clearance.

[0112] This invention, through sequencing, revealed that exosome treatment significantly reduced the expression levels of CCR5 at both the gene and protein levels in microglia. Figure 14 (bd), demonstrating that umbilical cord stem cell exosomes are associated with CCR5, and that CCR5 is also closely related to the inflammatory response of microglia. Figure 14 -a). Therefore, this invention aims to use umbilical cord stem cell exosomes as carriers to carry siCCR5 for the treatment of Alzheimer's disease.

[0113] (2) Establishment and detection of mouse models

[0114] (1) Grouping of animals

[0115] The experimental animals used were APP / PS1 transgenic mice, which carry five familially inherited AD mutation genes, including three APP mutation genes and two PS1 mutation genes. These mice exhibit human AD behavioral and pathological characteristics, such as amyloid protein deposition and overactivated glial cells in the hippocampus and cortical tissues. Since the APP / PS1 transgenic mice are heterozygous, male APP / PS1 transgenic mice were mated with female C57BL / 6 wild-type mice. Theoretically, the probability of obtaining APP / PS1 transgenic mice is one in four; therefore, genotyping of the offspring is necessary. In this study, PCR was used to detect the genotype of DNA from the tails of one-month-old mice using primers for both the APP and PSEN genes. Positive bands identified the transgenic model mice. (See attached image). Figure 15 .

[0116] As shown in Table 1, the experiment was divided into four groups: normal wild-type mice, AD disease group + PBS group, AD disease group + umbilical cord stem cell exosome group, and AD disease group + siCCR5 engineered umbilical cord stem cell exosome group, with six mice in each group. Exosomes were injected into the mice intranasally at a dose of 50 mg / kg, once a week, for one month. The differences before and after exosome injection were observed.

[0117] Table 1. Grouping and Treatment in In Vivo Experiments

[0118] Grouping number reagents dose Injection frequency time WT 5 physiological saline 15μL Twice a week 4 weeks AD 5 physiological saline 15μL Twice a week 4 weeks AD-Exos 5 hUCMSC-Exos 30 μg (15 μL) Twice a week 4 weeks AD-siCCR5Exos 5 hUCMSC-siCCR5Exos 30 μg (15 μL) Twice a week 4 weeks

[0119] (2) New Object Recognition Experiment

[0120] 1) Before conducting the test, get rid of the unfamiliarity with the mouse by petting it every day to avoid stimulating the mouse during the operation.

[0121] 2) Adaptation period: Mice move freely in the experimental device (without objects) for 10 minutes.

[0122] 3) Familiarization period: Place two identical objects (A and B, ensuring the objects are odorless and fixed in place) in the device, about 10 cm away from the side walls (to give the experimental animals space to explore).

[0123] 4) Place the mouse with its back to the object into the device from an equal distance away from the object, and use a camera and software to record the time the mouse spends exploring each object within 5 minutes (exploration of the object is defined as the mouse's mouth or nose touching the object or getting within about 2-3 cm of the object).

[0124] 5) After 24 hours, the test was conducted. One of the two identical objects was replaced with a different object and placed in the device (AC). The mouse was placed in the device from an equal distance away from the object with its back to the object. The camera equipment and software were used to record the time the mouse spent exploring each object within 5 minutes.

[0125] (3) New Location Recognition Experiment

[0126] In the training phase of the new object recognition experiment, two identical objects were placed on the same side. Now, in the new location recognition experiment, the training phase remains the same, but during the testing phase, the two objects are placed diagonally opposite each other. The mouse is placed in the device from an equidistant distance from the objects, with its back to them. A camera and software record the time the mouse spends exploring the objects at each location within 5 minutes. The results are as follows: Figure 16 As shown.

[0127] Figure 16The results showed that in the experiments on new object recognition and new location recognition, the AD model mice spent a relatively small proportion of time exploring new objects (new locations), roughly the same as the time spent exploring old objects (old locations). However, the Exos and siCCR5 Exos groups significantly increased the proportion of time spent exploring new objects (new locations), with the siCCR5 Exos group showing a more significant effect. This indicates that stem cell exosomes can significantly improve the memory ability of mice, and the engineered exosomes of siCCR5 have a better memory improvement effect.

[0128] (4) Morris's Water Maze Experiment

[0129] A. The mice need to be trained first:

[0130] 1) In water maze training, the platform should be located in the center of the pool, 1 cm above the water surface, so that the animals are aware of its presence. The water temperature should be 26℃.

[0131] 2) Each animal will undergo three consecutive trials. Place the animal on the platform for 20 seconds.

[0132] 3) The water maze has four quadrants. Place the animal in one of the quadrants. Lower the animal into the water by supporting it with your hand and gently placing it tail-first. Do not let the animal go head-first into the water to avoid stress.

[0133] 4) Let the animal swim to find the platform, for no more than 60 seconds. Initially, the animal may swim along the edge of the pool, looking for an exit. Eventually, the animal will learn to find the platform and climb onto it.

[0134] 5) Once the animal reaches the platform, stop timing and record the time. If it cannot find the platform within 60 seconds, record it as one minute. If the animal cannot reach the platform, do not pick it up. You can use a glass or plastic rod to guide the animal to the platform and let it stay on the platform for 15 seconds. Repeat the same procedure for the other two quadrants, in different directions for each trial.

[0135] 6) After the animals have completed all three trials, dry them with a towel. Repeat the three-trial training process for all animals in turn. Keep all animals facing the same direction and record their times.

[0136] B. After the animals have received five days of training, they can be prepared for a water maze test.

[0137] 1) Before starting the water maze experiment, fill the pool with tap water and heat it. Set the platform 1 cm below the water surface. Use skim milk, or 125 ml of non-toxic white temporary paint, or food coloring to make the water opaque.

[0138] 2) The lighting and water temperature should be the same as during training.

[0139] 3) Analyze the animal until it reaches the platform and record the time taken. If the animal does not reach the platform within 60 seconds, the handler will guide it to the platform, just as in training. Let the animal stay on the platform for 10 seconds, then dry it off and return it to the holding cage.

[0140] 4) Perform four trials on each animal, placing them in different quadrants, with the animal handler returning to the same designated location during each trial. The testing order should be: first trial for all animals, second trial for all animals, third trial for all animals, and so on. There should be at least two minutes between trials.

[0141] 5) The underwater platform was dismantled for testing. Each mouse swam in the pool at 60-second intervals, exploring the area previously occupied by the platform. Water maze software was used to record the exploration trajectory of each mouse during training and testing for subsequent analysis.

[0142] 6) Once all the exploratory experiments are complete, dry the animals and drain the pool.

[0143] See results Figure 17 .

[0144] In the mouse water maze experiment Figure 17 The results showed that on the third day of training, the escape latency of the WT group was significantly shortened, and they were able to quickly find the platform. On the fourth day of training, the escape latency of the Exos group was shortened; on the fifth day, the effect was even more pronounced in the siCCR5-Exos group. Figure 17 In the test trajectory of tc / b, the number of times APP / PS1 crossed the platform within 60s and the time spent in the target quadrant were significantly lower than WT. Both of these indicators increased in the Exos group, with the siCCR5-Exos group showing a more significant increase. Figure 17 China and Figure 17 The results showed no significant differences in swimming speed and total swimming distance among the groups of mice, indicating that the differences between mice were not affected by swimming ability. These results demonstrate that exosomes can effectively improve spatial memory in mice, and the engineered exosome siCCR5-Exos is more effective than ordinary exosomes.

[0145] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing engineered umbilical cord stem cell exosomes carrying siCCR5, characterized in that, Includes the following steps: (1) Preparation of liposome membrane: DLin-MC3-DMA was dissolved in an organic solvent, and cholesterol and DOPC were further dissolved to prepare a liposome solution; the organic solvent was removed by rotary evaporation, and the solution was dried under an inert gas environment to obtain a liposome membrane; (2) Preparation of engineered exosomes: The lipid membrane was dissolved with PBS, and after sonication, siCCR5 and umbilical cord mesenchymal stem cell exosomes were added to obtain a mixed solution. Under the condition of heat preservation, the solution was further filtered, squeezed and dialyzed to obtain engineered exosomes. In step (1), the organic solvent is a mixed solvent of chloroform and methanol in a volume ratio of 9:1; the molar ratio of DLin-MC3-DMA: cholesterol: DOPC is 25~34.5:35~65:1.5~40. In step (2), the mass ratio of lipid membrane to PBS during hydration and dissolution is 100:3; the mass ratio of siCCR5: umbilical cord mesenchymal stem cell exosomes: lipid membrane is 2:1:

50. In step (2), the filtering and extrusion step is as follows: The mixed solution was first squeezed through a filter with a pore size of 1.0 μm, then squeezed a second time through a filter with a pore size of 0.1 μm, and finally squeezed a third time through a filter with a pore size of 0.05 μm.

2. The method for preparing engineered umbilical cord stem cell exosomes carrying siCCR5 according to claim 1, characterized in that, In step (2), the temperature of the heat preservation is 40~50℃.

3. The method for preparing engineered umbilical cord stem cell exosomes carrying siCCR5 according to claim 1, characterized in that, The filters used in the three extrusions are polycarbonate filters, and the number of extrusions is 2 to 5 times each.

4. The method for preparing engineered umbilical cord stem cell exosomes carrying siCCR5 according to claim 1, characterized in that, In step (2), the dialysis procedure is as follows: Dialysis was performed overnight at 4°C using a dialysis cartridge with a pore size of 100 kDa, and the pH was adjusted to 7.2–7.

4.

5. An engineered exosome prepared by the method for preparing engineered umbilical cord stem cell exosomes carrying siCCR5 as described in any one of claims 1 to 4.

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