Stem cell exosome for overexpressing A beta degrading enzyme as well as preparation method and application of stem cell exosome

Abstract

The invention provides a stem cell exosome for overexpressing A beta degrading enzyme as well as a preparation method and application of the stem cell exosome. Specifically, the invention provides an application of the stem cell exosome overexpressing A beta degrading enzyme in preparation of drugs, and the drugs are used for at least one of the following: prevention, improvement or treatment of cognitive impairment related diseases; neuronal cell damage is improved; wherein the stem cells for overexpressing the A beta degrading enzyme are prepared through lentivirus transfection. According to the stem cell exosome overexpressing the A beta degrading enzyme, the content of the A beta degrading enzyme is obviously higher than that of the exosome prepared by a traditional method. Therefore, when the compound is prepared into a medicine for treating or preventing cognitive impairment related diseases, the medicine can be efficiently conveyed to the brain, and the concentration of the medicine in the brain is remarkably improved, so that the curative effect is enhanced, the dosage of the medicine can be reduced, the side effect is reduced, and the prognosis of brain diseases is effectively improved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a stem cell exosome overexpressing Aβ-degrading enzyme, and a preparation method and application thereof. Background Art

[0002] Take Alzheimer's disease (AD) as an example. AD is the most common neurodegenerative disease in the elderly, characterized by progressive memory loss and cognitive impairment. Neurofibrillary tangles and senile plaques are two histopathological hallmarks of AD. Senile plaques are formed by the abnormal aggregation and deposition of β-amyloid protein (Aβ). Therefore, it is widely accepted that the core mechanism of AD is Aβ deposition. The generation and clearance of Aβ in the normal human brain is a dynamic equilibrium. Disruption of this equilibrium leads to excessive Aβ deposition in the hippocampus, thus triggering AD. Recent studies have demonstrated that most Aβ is not cleared through transport into the vascular system, but rather is cleared from the body through degradation into small peptides by Aβ-degrading enzymes. Studies have shown that the main Aβ-degrading enzymes include insulin-degrading enzyme (IDE), neprilysin (NEP), endothelin-converting enzyme-1 (ECE-1), and endothelin-converting enzyme-2 (ECE-2).

[0003] Increasing the levels of pathogenic protein-degrading enzymes may be a potential clinical strategy for treating AD in fields such as biomedicine. The creation of stem cell exosomes that overexpress Aβ-degrading enzymes could overcome the inherent limitations of enzyme drugs and achieve the goal of treating brain diseases. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art to a certain extent. To this end, the present invention provides the use of stem cell exosomes that overexpress Aβ-degrading enzyme in drug preparation. Experiments have found that compared with loading Aβ-degrading enzyme into normal exosomes by ultrasound, the content of Aβ-degrading enzyme in exosomes derived from stem cells that overexpress Aβ-degrading enzyme is significantly higher. Therefore, using such exosomes to treat brain diseases can more efficiently deliver drugs to the brain, increase the concentration of drugs in the brain, and thus enhance the therapeutic effect. At the same time, it can also reduce drug dosage, reduce side effects, and help improve the prognosis of brain diseases.

[0005] Therefore, in its first aspect, the present invention proposes the use of stem cell exosomes overexpressing Aβ-degrading enzyme in the preparation of a drug for at least one of the following purposes: preventing, ameliorating, or treating diseases associated with cognitive impairment; or ameliorating neuronal cell damage. The stem cells overexpressing Aβ-degrading enzyme are prepared by lentiviral transfection. During experiments, the inventors unexpectedly discovered that the stem cell exosomes overexpressing Aβ-degrading enzyme prepared by the present invention contain significantly higher levels of Aβ-degrading enzyme than exosomes prepared by conventional methods. Therefore, when formulated into a drug for the treatment or prevention of diseases associated with cognitive impairment, the drug can be efficiently delivered to the brain, significantly increasing brain drug concentrations and thereby enhancing efficacy. It can also reduce drug dosage, minimize side effects, and effectively improve the prognosis of brain diseases. Furthermore, in vitro experiments have shown that stem cell exosomes overexpressing Aβ-degrading enzyme can promote neuronal cell growth, axonal growth in AD model cells, and ameliorate neuronal cell damage.

[0006] In the second aspect of the present invention, the present invention proposes a method for preparing stem cell exosomes that overexpress Aβ-degrading enzyme. According to an embodiment of the present invention, the method comprises: using a lentivirus that overexpresses Aβ-degrading enzyme to transfect stem cells to be treated, so that the stem cells overexpress Aβ-degrading enzyme; culturing the stem cells that overexpress Aβ-degrading enzyme; collecting the cell supernatant after the culturing treatment; and subjecting the cell supernatant to multiple centrifugation treatments to obtain the stem cell exosomes that overexpress Aβ-degrading enzyme. Therefore, compared with loading Aβ-degrading enzyme into normal exosomes by ultrasound, the Aβ-degrading enzyme-overexpressing stem cell exosomes prepared by the method of the present invention have a significantly higher content of Aβ-degrading enzyme, which can improve the bioavailability of the drug, reduce the drug dosage, and reduce side effects.

[0007] In a third aspect, the present invention provides stem cell exosomes that overexpress Aβ-degrading enzyme. According to an embodiment of the present invention, the stem cell exosomes that overexpress Aβ-degrading enzyme are prepared by the method described in the second aspect. Thus, the stem cell exosomes overexpressing Aβ-degrading enzyme prepared by the present invention have a significantly higher Aβ-degrading enzyme content than exosomes prepared by traditional methods. When used to treat or prevent diseases related to cognitive dysfunction, these exosomes can improve the bioavailability of drugs for brain diseases, reduce drug dosage, and reduce sexual side effects, thereby improving the prognosis of brain diseases.

[0008] In the fourth aspect of the present invention, the present invention proposes a method for improving neuronal cell damage in vitro. According to an embodiment of the present invention, the method comprises: co-culturing damaged neuronal cells with stem cell exosomes that overexpress Aβ degrading enzyme as described in the third aspect. During the experiment, the inventor unexpectedly discovered that stem cell exosomes that overexpress Aβ degrading enzyme can promote neuronal cell growth, while promoting axonal growth of AD model cells and improving neuronal cell damage. Therefore, the method of the present invention can be used to improve neuronal cell damage in vitro.

[0009] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0011] Figure 1 This is a schematic diagram of the lentiviral vector purchased from GeneCare Genetics;

[0012] Figure 2 The exosomes (hUC-MSC-EXO) derived from human umbilical cord mesenchymal stem cells (HUC-MSCs) overexpressing IDE according to Example 1 of the present invention are LV-IDE , referred to as "EXO LV-IDE ”) morphology and particle concentration characterization results, where:

[0013] A is for EXO LV-IDE Transmission electron microscopy results;

[0014] B is for EXO LV-IDE Nanoparticle tracking analysis technology test results;

[0015] Figure 3 EXO according to Example 1 of the present invention LV-IDE and the size and zeta potential characterization results of human umbilical cord mesenchymal stem cell exosomes (hUC-MSC-EXO, referred to as "EXO"), where:

[0016] A is the EXO measured by the particle size analyzer LV-IDE and the particle size results of EXO;

[0017] B is EXO measured by particle size analyzer LV-IDE and Zeta potential results of EXO;

[0018] Figure 4EXO is measured by the ELISA kit according to Example 1 of the present invention LV-IDE The relative levels of IDE contained in EXO;

[0019] Figure 5 EXO according to Example 2 of the present invention LV-IDE Comparison of IDE loading capacity between the two methods using ultrasound and EXO, where:

[0020] A is the band diagram of western blot experimental results;

[0021] B is the relative quantitative statistical graph of the gray value of the strip;

[0022] Figure 6 EXO according to Example 3 of the present invention LV-IDE The results of the in vitro therapeutic effect investigation of the drug system are shown in the figure, where:

[0023] A is the evaluation of neuronal cell survival rate of AD model cells;

[0024] B is the result of Aβ degradation at the cellular level in AD model;

[0025] C is a graph showing the effect on axonal growth of AD model cells;

[0026] Figure 7 EXO according to Example 4 of the present invention LV-IDE Results of the investigation of the drug system's brain accumulation ability, including:

[0027] A is the fluorescence imaging of organs in each experimental group;

[0028] B is the relative fluorescence quantitative statistical graph of the experimental group. DETAILED DESCRIPTION

[0029] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0030] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0031] The endpoints of the ranges and any values disclosed herein are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0032] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention, but not excluding other contents.

[0033] As used herein, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0034] In this article, the term "Transmission Electron Microscope (TEM) is a high-resolution microscope that uses electron beams instead of light beams, which can provide higher magnification and resolution than traditional optical microscopes. It is usually used to observe nanoscale samples such as viruses, organelles, and macromolecular complexes.

[0035] As used herein, the term “Nanoparticle Tracking Analysis (NTA)” refers to a biophysical method used to analyze and characterize the size and concentration of nanoparticles.

[0036] In an embodiment of the present invention, transmission electron microscopy and nanoparticle tracking analysis technology are used to characterize the morphology, particle concentration and size of exosomes, respectively.

[0037] In this article, the term "dynamic light scattering" refers to a physical characterization method that analyzes the motion characteristics of particles in solution by measuring the fluctuation of light intensity over time based on the scattering of light by particles, thereby determining the size distribution and other physicochemical properties of the particles (such as zeta potential).

[0038] In this article, the term "exosomes (abbreviated as "EXO") refers to a type of extracellular vesicle with a diameter of approximately 30 to 150 nm, which can be secreted by almost all types of cells (including cells in the central nervous system) under physiological or pathological conditions. Exosomes can directly act on receptor cells to play physiological roles such as intercellular substance transport and signal transduction. The differences in substances contained in exosomes from different cells mainly depend on the parent cells. Therefore, exosomes from different sources have different functions. Transmission electron microscopy can provide information about the morphology, size and structure of exosomes, which is crucial for studying the physical properties of exosomes and evaluating their potential as drug carriers. Nanoparticle tracking analysis technology can accurately measure the hydrodynamic diameter of exosomes, which helps to understand the size distribution and uniformity of exosomes; it can also quantitatively analyze the particle concentration of exosomes, providing basic data for studying their biological functions and potential applications.

[0039] In this article, the term "EXO LV-IDE " indicates exosomes derived from human umbilical cord mesenchymal stem cells (HUC-MSCs) that overexpress IDE (hUC-MSC-EXO LV-IDE , referred to as "EXO LV-IDE ”), which is equivalent to “stem cell exosomes overexpressing IDE”.

[0040] The present invention proposes the use of stem cell exosomes overexpressing Aβ-degrading enzyme in the preparation of drugs, a method for preparing stem cell exosomes overexpressing Aβ-degrading enzyme, stem cell exosomes overexpressing Aβ-degrading enzyme, and a method for improving neuronal cell damage in vitro, each of which will be described in detail below.

[0041] Use of stem cell exosomes overexpressing Aβ-degrading enzyme in the preparation of drugs

[0042] In its first aspect, the present invention proposes the use of stem cell exosomes overexpressing Aβ-degrading enzyme in the preparation of a drug for at least one of the following purposes: preventing, ameliorating, or treating diseases associated with cognitive impairment; or ameliorating neuronal cell damage. The stem cells overexpressing Aβ-degrading enzyme are prepared via lentiviral transfection. During experiments, the inventors unexpectedly discovered that the stem cell exosomes overexpressing Aβ-degrading enzyme prepared by the present invention contain significantly higher levels of Aβ-degrading enzyme than exosomes prepared by conventional methods. Therefore, when formulated into a drug for the treatment or prevention of diseases associated with cognitive impairment, the drug can be efficiently delivered to the brain, significantly increasing brain drug concentrations and thereby enhancing efficacy. It can also reduce drug dosage, minimize side effects, and effectively improve the prognosis of brain diseases. Furthermore, in vitro experiments have shown that stem cell exosomes overexpressing Aβ-degrading enzyme can promote neuronal cell growth, axonal growth in AD model cells, and ameliorate neuronal cell damage.

[0043] In some embodiments of the present invention, the administration of the drug includes: nasal administration, oral administration, and intravenous injection.

[0044] In some embodiments of the present invention, the drug is administered via nasal route. The inventors discovered during experiments that nasal administration helps bypass the blood-brain barrier, allowing for more efficient drug delivery to the brain and increasing drug concentrations within the brain, thereby enhancing therapeutic efficacy. It also reduces the distribution of Aβ-degrading enzymes to non-target tissues, improving therapeutic specificity. Furthermore, bypassing the blood-brain barrier can improve drug bioavailability, reduce drug dosage, and mitigate side effects, ultimately improving the prognosis of brain diseases.

[0045] In some embodiments of the present invention, the Aβ-degrading enzymes include, but are not limited to, insulin-degrading enzyme (IDE), neprilysin (NEP), endothelin-converting enzyme-1 (ECE-1), and endothelin-converting enzyme-2 (ECE-2). Thus, these Aβ-degrading enzymes can effectively reduce Aβ levels in the brain, delaying or preventing the formation of Aβ deposits and related cellular pathological changes.

[0046] In the present invention, the term "stem cell (stem cell)" as a relatively undeveloped undifferentiated cell refers to a cell with the ability to differentiate into the cells of a specific tissue. Based on differentiation ability, stem cells can be divided into pluripotent stem cells (pluripotent stem cell), pluripotent stem cells (multipotent stem cell) or unipotent stem cells (unipotent stem cell). In addition, according to the cell of origin, the above-mentioned stem cells can be divided into embryonic stem cells (embryonic stem cell), adult stem cells (adult stem cell) or induced pluripotent stem cells (induced pluripotent stem cell, iPSC) prepared from human somatic cells. Specifically, the stem cell of the present invention can be an adult stem cell. The above-mentioned term "adult stem cell" refers to an undifferentiated cell that can differentiate into a desired cell and can self-renew (self-renew) that is present in an adult tissue or organ. As an example, the stem cell of the present invention can be an amniotic membrane-derived adult stem cell.

[0047] In some embodiments of the present invention, the stem cells are derived from, but are not limited to, umbilical cord, bone marrow, embryo, placenta, fat, bone, cartilage, periodontium, synovium, muscle, lung, liver, and pancreas.

[0048] It should be noted that, in the present invention, when stem cells are derived from human umbilical cord, embryo or placenta, the human umbilical cord, embryo or placenta referred to is within 14 days of fertilization and has not undergone in vivo development.

[0049] In some embodiments of the present invention, the stem cells are mesenchymal stem cells, thereby improving the brain targeting of stem cell exosomes that overexpress Aβ degrading enzyme.

[0050] According to an embodiment of the present invention, the stem cells include but are not limited to: umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, dental pulp mesenchymal stem cells, placental mesenchymal stem cells, amniotic membrane mesenchymal stem cells, synovial mesenchymal stem cells, and thymus mesenchymal stem cells.

[0051] It should be noted that, in the present invention, the above-mentioned mesenchymal stem cells can generally be obtained commercially or by methods known in the art. The purchase or acquisition of the above-mentioned mesenchymal stem cells, especially human umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells (hBMSCs), placental mesenchymal stem cells, and amniotic membrane mesenchymal stem cells, should be carried out in compliance with local laws and regulations, including regulations on cell therapy, import and export of biological materials, ethics and privacy protection. When the exosomes described in the present invention are obtained from umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, placental mesenchymal stem cells, and amniotic membrane mesenchymal stem cells, these stem cells should also be within 14 days of fertilization without undergoing in vivo development.

[0052] According to an embodiment of the present invention, the stem cells are derived from animals including but not limited to mice, rats, rabbits, dogs, pigs, and primates.

[0053] In some embodiments of the invention, the primate is a human primate.

[0054] In some embodiments of the present invention, the stem cells are derived from human umbilical cord mesenchymal stem cells.

[0055] In some embodiments of the present invention, the drug may further include a pharmaceutically acceptable carrier or excipient.

[0056] In the present invention, the term "pharmaceutically acceptable" means that the substance or composition must be chemically and / or toxicologically compatible with the other ingredients of the formulation and / or the mammal to be treated therewith. Preferably, the "pharmaceutically acceptable" herein means approved by federal regulatory agencies or national governments or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia for use in animals, particularly humans.

[0057] As used herein, the term "pharmaceutically acceptable carrier" includes any solvent, pharmaceutical stabilizer, or combination thereof, known to those skilled in the art. Except where any conventional carrier is incompatible with the active ingredient, its use in therapeutic or pharmaceutical compositions is encompassed.

[0058] In the present invention, the term "pharmaceutically acceptable excipient" includes any solvent suitable for the specific target dosage form. Except to the extent that any conventional excipient is incompatible with the exosomes disclosed herein, such as by producing any adverse biological effects or interacting in a deleterious manner with any other component of the pharmaceutically acceptable composition, their use is also contemplated by the present invention.

[0059] In some embodiments of the present invention, the cognitive dysfunction may include all known diseases caused by cognitive dysfunction, or diseases in which cognitive dysfunction occurs due to the onset of the disease. Specifically, the cognitive dysfunction may be a disease in which the expression or aggregation level of β-amyloid protein is higher than normal or there is a risk of increase. For example, the cognitive dysfunction may be dementia, Alzheimer's disease, senile dementia, early senile dementia, Parkinson's disease, Huntington's disease, mild cognitive impairment, cerebral amyloid angiopathy, Down syndrome, amyloid stroke, vascular stroke, systemic amyloidosis, Dutch amyloidosis, Niemann-Pick disease, multiple sclerosis, Lewy body dementia, Creutzfeldt-Jakob disease or frontotemporal dementia.

[0060] In the present invention, "prevention" and "prevent" are used interchangeably. These terms refer to an approach to achieving a beneficial or desired result, including but not limited to a prophylactic benefit. To achieve a "prophylactic benefit," stem cell exosomes overexpressing Aβ-degrading enzymes, or products containing the same, can be administered to a subject at risk for a particular disease, or to a subject reporting one or more physiological symptoms of a disease, even though a diagnosis of the disease may not have yet been made.

[0061] In the present invention, the terms "treat" and "alleviate" all refer to the use of drugs to obtain the desired pharmacological and / or physiological effects. The effect can be preventive in terms of completely or partially preventing the disease or its symptoms, and / or can be therapeutic in terms of partially or completely curing the disease and / or the adverse effects caused by the disease. "Treatment" as used herein covers diseases in mammals, especially humans, including: (a) preventing the occurrence of diseases or conditions in individuals who are susceptible to the disease but have not yet been diagnosed with the disease; (b) inhibiting the disease, such as blocking the development of the disease; or (c) alleviating the disease, such as alleviating the symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a drug or compound to an individual to treat, cure, alleviate, improve, reduce or inhibit the individual's disease, including but not limited to administering a drug containing a compound described herein to an individual in need.

[0062] In some embodiments of the present invention, the stem cells overexpressing Aβ-degrading enzyme are prepared by transfecting the stem cells to be treated with a lentivirus that overexpresses Aβ-degrading enzyme, thereby overexpressing the Aβ-degrading enzyme in the stem cells. Thus, stem cells overexpressing Aβ-degrading enzyme prepared by lentiviral transfection have high infection efficiency and can maintain long-term stable gene expression during cell passage.

[0063] In some embodiments of the present invention, the transfection concentration of the lentivirus is 2×10 6-8 TU / mL. For example, it can be 2×10 6 TU / mL, 2×10 6.5TU / mL, 2×10 7 TU / mL, 2×10 7.5 TU / mL, 2×10 8 TU / mL, etc., or may be within a range consisting of any of the above values.

[0064] In some embodiments of the present invention, the transfection time is 10-14 hours, for example, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, etc., or any range thereof.

[0065] Preparation method of stem cell exosomes overexpressing Aβ degrading enzyme

[0066] In the second aspect of the present invention, the present invention proposes a method for preparing stem cell exosomes that overexpress Aβ-degrading enzyme. According to an embodiment of the present invention, the method comprises: using a lentivirus that overexpresses Aβ-degrading enzyme to transfect stem cells to be treated, so that the stem cells overexpress Aβ-degrading enzyme; culturing the stem cells that overexpress Aβ-degrading enzyme; collecting the cell supernatant after the culturing treatment; and subjecting the cell supernatant to multiple centrifugation treatments to obtain the stem cell exosomes that overexpress Aβ-degrading enzyme. Therefore, compared with loading Aβ-degrading enzyme into normal exosomes by ultrasound, the Aβ-degrading enzyme-overexpressing stem cell exosomes prepared by the method of the present invention have a significantly higher content of Aβ-degrading enzyme, which can improve the bioavailability of the drug, reduce the drug dosage, and reduce side effects.

[0067] In this article, the term "loading rate" refers to the loading efficiency of the target molecule in the exosome. The loading rate reflects the utilization efficiency of the carrier and the encapsulation efficiency of the target molecule.

[0068] In some embodiments of the present invention, the transfection concentration of the lentivirus is 2×10 6-8 TU / mL. For example, it can be 2×10 6 TU / mL, 2×10 6.5 TU / mL, 2×10 7 TU / mL, 2×10 7.5 TU / mL, 2×10 8 TU / mL, etc., or may be within a range consisting of any of the above values.

[0069] In some embodiments of the present invention, the transfection time is 10-14 hours, for example, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, etc., or any range thereof.

[0070] Stem cell exosomes overexpressing Aβ-degrading enzymes

[0071] In a third aspect, the present invention provides stem cell exosomes that overexpress Aβ-degrading enzyme. According to an embodiment of the present invention, the stem cell exosomes that overexpress Aβ-degrading enzyme are prepared by the method described in the second aspect. Thus, the stem cell exosomes that overexpress Aβ-degrading enzyme prepared by the present invention have a significantly higher Aβ-degrading enzyme content than exosomes prepared by traditional methods. When used to treat or prevent diseases related to cognitive dysfunction, these exosomes can improve the bioavailability of drugs for brain diseases, reduce drug dosage, and reduce side effects, thereby improving the prognosis of brain diseases.

[0072] Method for improving neuronal cell damage in vitro

[0073] In the fourth aspect of the present invention, the present invention proposes a method for improving neuronal cell damage in vitro. According to an embodiment of the present invention, the method comprises: co-culturing damaged neuronal cells with stem cell exosomes that overexpress Aβ degrading enzyme as described in the third aspect. During the experiment, the inventor unexpectedly discovered that stem cell exosomes that overexpress Aβ degrading enzyme can promote neuronal cell growth, while promoting axonal growth of AD model cells and improving neuronal cell damage. Therefore, the method of the present invention can be used to improve neuronal cell damage in vitro.

[0074] The following embodiments of the present invention are described in detail. The embodiments described below are exemplary and are only used to explain the present invention, and are not to be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, the techniques or conditions described in the literature in this field or the product specifications are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be obtained commercially.

[0075] Example 1: Exosomes of human umbilical cord mesenchymal stem cells overexpressing IDE (EXO LV-IDE )

[0076] In this example, primary mesenchymal stem cells extracted from human umbilical cord (purchased) were cultured. When the cells grew to 70-80% confluence, they were digested and passaged using 0.25% trypsin at a passage ratio of 1:2.

[0077] Lentivirus was purchased from GeneCare Gene Co., Ltd. (the packaging vector of lentivirus is as follows Figure 1 As shown), 10 μL was taken with a titer of 2×10 9TU / mL lentivirus, mixed with 25μL of HiTransG P viral transfection reagent, was co-cultured with 2-3 generations of human umbilical cord mesenchymal stem cells grown to 70-80% confluency to construct human umbilical cord mesenchymal stem cells overexpressing insulin degrading enzyme (IDE). After 12 hours of transfection, fresh complete medium (human umbilical cord mesenchymal stem cell complete medium, purchased from Purosai) was replaced and cultured for 72 hours. The cells were then cultured in a culture medium containing 1.0μg / mL puromycin. After 48 hours of culture and screening, all normal human umbilical cord mesenchymal stem cells (empty cell group) co-cultured with puromycin died, and the remaining human umbilical cord mesenchymal stem cells in the lentivirus-infected group were positive cells. Subsequently, the puromycin concentration was reduced to 0.25μg / mL, and the infected cells were screened and amplified. The following experiments were carried out using these screened cells.

[0078] When the cell confluence reached 60-70%, the complete culture medium was replaced with human umbilical cord mesenchymal stem cell complete culture medium containing 10% exosome-free serum and cultured for another 48 hours. The culture supernatant of human umbilical cord mesenchymal stem cells overexpressing IDE and normally cultured human umbilical cord mesenchymal stem cells was collected and subjected to gradient centrifugation at 300g for 10 minutes, 3000g for 25 minutes, and 10000g for 1 hour. The supernatant was concentrated by ultrafiltration (molecular weight cutoff of 100 kDa) to obtain the exosome solution.

[0079] The two exosome solutions were characterized by transmission electron microscopy and nanoparticle tracking analysis, as well as dynamic light scattering and Zeta potential. Figure 2 and Figure 3 As shown, the particles in the exosome solution of human umbilical cord mesenchymal stem cells overexpressing IDE are spherical vesicle-like structures, which are consistent with the extracellular vesicle structure of exosomes ( Figure 2 A); Under the condition of 500-fold dilution, the particle concentration is 2.1×10 10 Particles / mL, with a particle size of 40-150 nm, which is consistent with the particle size of exosomes ( Figure 2 B), indicating that the drug system prepared by the present invention is nanoscale and has a small particle size, making it easier to penetrate biological barriers, such as the blood-brain barrier, which is particularly important for the treatment of brain diseases. The hydrodynamic diameter of the exosome solution of human umbilical cord mesenchymal stem cells overexpressing IDE was measured by a particle size analyzer to be 111.35±16.15nm, which is consistent with the size of the exosome solution of normally cultured human umbilical cord mesenchymal stem cells ( Figure 3 A), indicating that the drug system has uniform particle size and is suitable for in vivo application, especially targeted delivery through the vascular system; the zeta potential is -12.95±4.75mV, which is consistent with the zeta potential of the exosome solution of normally cultured human umbilical cord mesenchymal stem cells ( Figure 3 B), thus, it is beneficial to in vivo biocompatibility, reduces binding to proteins in the body, and can form stable dispersions in physiological environments, which helps ensure consistent and predictable drug delivery.

[0080] The exosomes of human umbilical cord mesenchymal stem cells overexpressing IDE (EXO LV-IDE ) and the IDE levels in the exosomes (EXO) of human umbilical cord mesenchymal stem cells cultured normally. Figure 4 As shown, EXO LV-IDE The IDE level in EXO is 114.8% higher than that in normal EXO.

[0081] Example 2: Comparison of Exosomes Loaded with IDE Prepared by Different Methods

[0082] In order to explore the exosomes (EXO) obtained from human umbilical cord mesenchymal stem cells overexpressing IDE LV-IDE The difference in IDE loading rates between the two methods of in vitro IDE loading was investigated. The inventors used normal exosomes with the same particle concentration to load IDE in vitro using ultrasound. The specific process of ultrasound loading IDE is as follows: EXO is mixed with an excess of IDE, i.e., 1.1×10 10 Particles EXO were mixed with 10 μg IDE protein, and the mixture was sonicated under the following conditions: ultrasonic temperature 4°C, power 90 kW, and time 0.5 h. After the sonication, the solution was ultrafiltered and concentrated using an ultrafiltration centrifuge tube (molecular weight cutoff 100 kDa) to remove free IDE and prepare the EXO-IDE drug system. LV-IDE Lysis buffer (1% protease inhibitor cocktail and 1% PMSF were added to the Beyotime western blot lysis buffer) was added to the EXO-IDE drug system, and the cells were placed at 4°C for 0.5 hours. The loading amount of IDE was then detected using a western blot experiment.

[0083] The experimental results are as follows Figure 5 As shown, contrary to the inventors' expectations, the EXO prepared by overexpression LV-IDE The IDE level in the drug system increased by 48% compared with the IDE level in the EXO-IDE drug system prepared by ultrasonic method. LV-IDE The level of IDE has been improved endogenously, with high efficiency and a more stable system.

[0084] Example 3: EXO LV-IDE Study on the therapeutic effect of drug systems in vitro

[0085] In order to verify the EXO LV-IDE The therapeutic effect of the drug system was tested in vitro. The specific process is as follows:

[0086] EXO was detected using CCK8 assay LV-IDE and EXO LV-APP The cell survival rate of SH-SY5Y cells (SH-SY5Y cells overexpressing amyloid precursor protein (APP) constructed by lentiviral transfection, which are AD model cells) (the concentration of EXO was uniformly set at 1.0×10 8 Particles / mL).

[0087] A Transwell double-layer cell barrier model was constructed to simulate the nasal mucosa-brain barrier in vitro. The upper layer was HNEpC (human nasal mucosal cells) and the lower layer was LV-APP SH-SY5Y cells were cultured for 48 hours and then grown in HNEpC- LV-APP EXO or EXO was added to the upper culture medium of the SH-SY5Y bilayer model. LV-IDE (The concentration of EXO is uniformly set to 1.0×10 8 Particles / mL), and the extracellular Aβ level was detected by ELISA after 24 hours of culture.

[0088] β3-Tubulin (Tuj1) is a major component of neuronal axonal microtubules and plays a key role in proper axon guidance, maturation and maintenance. It is mainly used to mark the neuronal cytoskeleton. LV-APP In SH-SY5Y cells, Tuj1 was stained with immunofluorescence, and the changes in axons were observed using a laser confocal fluorescence microscope.

[0089] The experimental results are as follows Figure 6 As shown, by applying the human umbilical cord mesenchymal stem cell exosomes (EXO LV-IDE ), at 1.0×10 8 Particles / mL of exosomes can promote neuronal cell growth ( Figure 6 A); EXO LV-IDE It can penetrate the simulated nasal mucosal barrier and reduce the extracellular Aβ level of neuronal cells ( Figure 6 B); It can promote the axonal growth of AD model cells and improve neuronal cell damage. Unexpectedly, EXO itself also has an improving effect on the axonal growth of neuronal cells, indicating that EXO LV-IDE The drug system realizes the synergistic therapeutic effect of EXO and IDE ( Figure 6C) Compared to the simple EXO, EXO LV-IDE The enzymatic activity of IDE can be better exerted after passing through tissue barriers (such as the nasal mucosal barrier).

[0090] Example 4: EXO LV-IDE Research on drug delivery methods

[0091] In order to make EXO LV-IDE The drug system can better exert the therapeutic effect. The inventors explored the EXO LV -IDE The optimal administration method of the drug system is as follows:

[0092] EXO was labeled with PKH26 dye for tracking, yielding PKH26-EXO LV-IDE Drug system.

[0093] 8-month-old APP / PS1 transgenic mice (AD mice) with a genetic background of C57BL / 6J were selected and randomly divided into 3 groups, 5 mice in each group. In the first group, APP / PS1 mice were orally administered with PKH26-EXO. LV-IDE (2.1×10 11 Particles / mL, 200 μL), and the second group was intravenously injected with PKH26-EXO at the same particle concentration into APP / PS1 mice. LV-IDE (2.1×10 11 Particles / mL, 200 μL), and the third group was nasally administered with the same particle concentration of PKH26-EXO to APP / PS1 mice. LV -IDE (2.1×10 12 Particles / mL, 20 μL), and 6 hours later, the PKH26 fluorescence intensity in the mouse brain was detected by fluorescence imaging.

[0094] The experimental results are as follows Figure 7 The results showed that nasal administration significantly increased EXO compared with oral and intravenous administration. LV-IDE Brain accumulation of Figure 7 A); Further fluorescence quantitative statistical analysis of each experimental group showed that compared with oral and intravenous administration, the brain penetration of nasal administration was increased by 2.40 times and 1.42 times, respectively, and the brain penetration efficiency was significantly enhanced ( Figure 7 B).

[0095] The above results show that EXO LV-IDE The drug system is administered through the nose, EXO LV-IDEIt can deliver drugs to the brain more effectively, increase the concentration of drugs in the brain, and thus enhance the therapeutic effect; enhanced brain accumulation also helps to reduce the distribution of drugs to non-target tissues and improve the specificity of treatment; due to its ability to bypass the blood-brain barrier, EXO LV-IDE It can also improve the bioavailability of brain disease drugs, reduce drug dosage, reduce side effects, and help improve the prognosis of brain diseases.

[0096] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0097] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. Use of stem cell exosomes overexpressing Aβ-degrading enzyme in the preparation of a medicament for at least one of the following: Prevent, improve or treat diseases related to cognitive impairment; Improve neuronal cell damage; among them, The stem cells overexpressing Aβ-degrading enzyme are prepared by lentiviral transfection.

2. The use according to claim 1, characterized in that The drug can be administered via nasal administration, oral administration, or intravenous injection.

3. The use according to claim 2, characterized in that The Aβ degrading enzymes include but are not limited to: insulin degrading enzyme, enkephalinase, endothelin converting enzyme-1, and endothelin converting enzyme-2.

4. The use according to claim 3, characterized in that The stem cells are derived from, but not limited to, umbilical cord, bone marrow, embryo, placenta, fat, bone, cartilage, periodontal tissue, synovium, muscle, lung, liver, and pancreas; Optionally, the stem cells are mesenchymal stem cells; Optionally, the stem cells include but are not limited to: umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, dental pulp mesenchymal stem cells, placental mesenchymal stem cells, amniotic membrane mesenchymal stem cells, synovial membrane mesenchymal stem cells, and thymus mesenchymal stem cells; Optionally, the stem cells are derived from, but not limited to, mice, rats, rabbits, dogs, pigs, and primates; further, the stem cells are human umbilical cord mesenchymal stem cells.

5. The use according to claim 4, characterized in that The diseases associated with cognitive dysfunction include at least one of dementia, Alzheimer's disease, senile dementia, early senile dementia, Parkinson's disease, Huntington's disease, mild cognitive impairment, cerebral amyloid angiopathy, Down syndrome, amyloid protein stroke, vascular stroke, systemic amyloidosis, Dutch amyloidosis, Niemann-Pick disease, multiple sclerosis, Lewy body dementia, Creutzfeldt-Jakob disease, and frontotemporal dementia.

6. The use according to claim 5, characterized in that The stem cells overexpressing Aβ degrading enzyme are prepared by the following method: The stem cells to be treated are transfected with a lentivirus that overexpresses Aβ-degrading enzyme, so that the Aβ-degrading enzyme is overexpressed in the stem cells.

7. The use according to claim 6, characterized in that The transfection concentration of the lentivirus was 2×10 6-8 TU / mL; Optionally, the transfection time is 10-14 hours.

8. A method for preparing stem cell exosomes that overexpress Aβ-degrading enzyme, characterized in that: include: transfecting the stem cells to be treated with a lentivirus that overexpresses Aβ-degrading enzyme, so that the Aβ-degrading enzyme is overexpressed in the stem cells; The stem cells that overexpress Aβ-degrading enzyme are cultured; collecting the cell supernatant after the culture treatment; The cell supernatant is centrifuged multiple times to obtain the stem cell exosomes that overexpress Aβ degrading enzyme.

9. A stem cell exosome overexpressing Aβ degrading enzyme, characterized in that: The method according to claim 8 is used for preparation.

10. A method for improving neuronal cell damage in vitro, characterized in that: include: The damaged neuronal cells are co-cultured with the stem cell exosomes overexpressing Aβ-degrading enzyme according to claim 9.

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