Pharmaceutical composition containing stem cell extracellular vesicles and its application in the treatment of respiratory inflammation

Abstract

The present invention provides a pharmaceutical composition containing stem cell vesicles and its use in treating acute and chronic respiratory inflammation. Specifically, the present invention provides a pharmaceutical composition with key active substances in the contents of stem cell vesicles as the main effective ingredients and a preparation method thereof, as well as the use of the pharmaceutical composition in treating acute respiratory inflammation and chronic obstructive pulmonary disease (COPD) and bronchial asthma. When the pharmaceutical composition preparation is administered through the respiratory tract, it can be effectively distributed in the small bronchi and alveolar parts of the lungs. Compared with conventional drugs or stem cell treatments, the main active ingredients of the pharmaceutical composition are easily taken up by small bronchial epithelial cells, alveolar epithelial cells, and inflammatory cells in the airways, thereby producing significant effects such as inhibiting airway inflammatory reactions, repairing airway damage, and improving airway tension, thereby effectively preventing or treating respiratory inflammation.

Description

Technical Field

[0001] The present invention relates to the field of stem cell therapeutic drugs, and in particular to a pharmaceutical composition of extracellular vesicles secreted by stem cells and an application thereof in the treatment of acute and chronic respiratory inflammation. Background Art

[0002] Lung inflammation is the body's natural response to injury. It helps the body remove pathogens, irritants and damaged cells, and initiates tissue repair processes. Acute and chronic lung inflammation is common in different respiratory diseases, such as asthma, cystic fibrosis, acute respiratory distress syndrome and chronic obstructive pulmonary disease (COPD). Among them, asthma and COPD are common chronic diseases in the world, affecting more than 300 million people worldwide and causing a large number of deaths each year. Currently, the morbidity and mortality of asthma and COPD are still increasing worldwide.

[0003] Asthma is a heterogeneous chronic airway inflammatory disease characterized by reversible airway obstruction. Its pathological features include airway hyperresponsiveness, infiltration of a large number of inflammatory cells in the bronchi and surrounding tissues, and excessive airway narrowing due to remodeling. COPD is a chronic airway inflammatory disease with incompletely reversible airflow limitation. Its pathological features are mainly inflammatory cell infiltration of airway tissue, airway remodeling, and alveolar damage. Without proper treatment and control, these two diseases may cause changes in the patient's airway, leading to irreversible alveolar damage and permanent pulmonary fibrosis, ultimately leading to loss of lung function and reduced responsiveness to therapeutic drugs.

[0004] At present, although many therapeutic drugs have been developed for the above-mentioned diseases, the course of the disease cannot be fully controlled. Conventional drugs for the treatment of asthma are mainly some drugs that relieve and control symptoms. Among them, relief drugs can quickly relieve bronchospasm and relieve asthma symptoms, but they cannot reverse the disease process, and patients usually develop tolerance and require higher doses to control symptoms. Control drugs are mainly some corticosteroids and leukotriene antagonists with anti-inflammatory effects, which need to be taken for a long time, so the side effects are large and cannot restore all changes related to asthma. In addition, the existing humanized antibody drug Omalizumab is only suitable for the treatment of severe asthma characterized by eosinophil infiltration, but is not suitable for the treatment of asthma characterized by neutrophil infiltration. Moreover, the therapeutic application of this antibody drug in some children with asthma is still limited. In addition, the drugs for the treatment of COPD are mainly some hormone drugs and bronchodilators. However, patients who use these drugs for a long time will also experience many side effects, such as tachycardia and hypertension.

[0005] Therefore, in order to avoid the disadvantages of long-term treatment with the above-mentioned conventional drugs, control the course of disease in severe patients, and improve the quality of life of patients, it is urgent to find a new treatment model to improve the current treatment methods. Summary of the invention

[0006] The purpose of the present invention is to provide a method for preparing a pharmaceutical composition with key active substances in extracellular vesicles of stem cells as main effective ingredients and its application in the treatment of acute and chronic respiratory inflammation.

[0007] In a first aspect of the present invention, a stem cell extracellular vesicle preparation is provided, wherein the particle size of the extracellular vesicle is 30-200 nm, preferably 50-150 nm, and more preferably 80-120 nm; and the extracellular vesicle contains bioactive factors related to anti-inflammatory or tissue damage repair, the bioactive factors including matrix protein metalloenzyme 1 (MMP-1) and hepatocyte growth factor (HGF), wherein the content of MMP-1 in the preparation is 1500-2000 pg / ml, and the content of HGF in the preparation is 5500-6000 pg / ml.

[0008] In another preferred embodiment, the stem cell extracellular vesicle preparation contains 1×10 8 -5×10 8 stem cell extracellular vesicles / ml.

[0009] In another preferred embodiment, the stem cell extracellular vesicles are derived from the supernatant collected during the in vitro culture of human stem cells.

[0010] In another preferred embodiment, the stem cells are selected from the group consisting of human umbilical cord blood-derived stem cells, human peripheral blood-derived stem cells, human umbilical cord mesenchymal stem cells, human placental mesenchymal stem cells, human adipose mesenchymal stem cells, human bone marrow-derived stem cells, or a combination thereof.

[0011] In another preferred embodiment, the stem cells are human adipose-derived mesenchymal stem cells.

[0012] In another preferred embodiment, the bioactive factors related to anti-inflammation or tissue damage repair are selected from the following group: MMP-1, HGF, vascular endothelial growth factor (VEGF), or a combination thereof.

[0013] In another preferred embodiment, the bioactive factor related to anti-inflammation or tissue damage repair is the bioactive factor produced by the human stem cells.

[0014] In another preferred embodiment, the bioactive factors related to anti-inflammatory or tissue damage repair include bioactive factors paracrine-produced by stem cells cultured in a natural state, bioactive factors expressed by stem cells modified with exogenous genes, or a combination thereof.

[0015] In another preferred embodiment, the stem cell extracellular vesicles have a complete lipid bilayer membrane structure and contain the following proteins: CD9, CD63, CD81, TSG101 and HSP70; and do not contain or substantially do not contain protein CANX.

[0016] In another preferred embodiment, the membrane structure surface of the stem cell extracellular vesicle contains CD9, CD63, and CD81, and the membrane structure inside contains TSG101 and HSP70.

[0017] In another preferred embodiment, the membrane structure of the stem cell extracellular vesicles also contains some non-coding functional DNA and RNA.

[0018] In another preferred embodiment, the RNA includes miRNA, tRNA, rRNA, snoRNA and snRNA.

[0019] In another preferred embodiment, the length of the RNA is 17 to 100 nucleotides.

[0020] In another preferred embodiment, the non-coding functional DNA and RNA include non-coding functional DNA and RNA molecules produced by stem cells cultured in a natural state, non-coding functional DNA and RNA molecules expressed by genetically engineered stem cells, non-coding functional DNA and RNA molecules transduced into stem cells after chemical synthesis, or a combination thereof.

[0021] In another preferred embodiment, the RNA is selected from the following group: hsa-let-7b-5p, hsa-let-7a-5p, hsa-miR-126-5p, hsa-miR-151a-3p, hsa-miR-1246, hsa-miR-21-5p, hsa-miR-146a-5p, hsa-let-7b-3p, has-miR-1290, has-miR-23a-3p, has-let-7d-3p or a combination thereof.

[0022] In another preferred embodiment, the cells used to produce the stem cell extracellular vesicles include cells from the following sources:

[0023] (a) Stem cells directly isolated and purified from human tissues;

[0024] (b) Cells obtained by directly isolating and purifying stem cells from human tissues and then proliferating these stem cells through minimal manipulation in a GMP laboratory;

[0025] (c) Cells obtained by specific gene modification, specific gene editing, specific gene transduction, or introduction of specific microRNA (miRNA) in a GMP laboratory;

[0026] (d) Cells obtained by pretreatment under special culture conditions in a GMP laboratory.

[0027] In another preferred embodiment, the cells used to produce the stem cell extracellular vesicles include primary cells and passaged cells with a passage number of 1-10.

[0028] In another preferred embodiment, the cells used to produce the stem cell extracellular vesicles include genetically unmanipulated cells and genetically manipulated cells.

[0029] In another preferred embodiment, the gene manipulation includes gene editing, gene introduction, gene knock-down, gene knock-out, or a combination thereof.

[0030] In another preferred embodiment, the stem cell extracellular vesicles have the following characteristics: they are prepared by polyethylene glycol (PEG) precipitation method, the particle size of the extracellular vesicles is concentrated between 80-120 nm, and the particle size is smaller and uniform.

[0031] In another preferred embodiment, the polyethylene glycol (PEG) precipitation includes the steps of: using PEG 3000-9000, configuring 8%-20% PEG mother solution with PBS, filtering and sterilizing (such as filtering with a 0.22μm filter), adding it to the treated conditioned medium (the conditioned medium is obtained by differential centrifugation and filtration of the culture supernatant of human adipose mesenchymal stem cells) in a certain ratio (such as about 1:1 volume ratio), and incubating at 4°C overnight; then centrifuging at 4°C, 3,000-5000g for 30-60 minutes, discarding the supernatant, adding pre-cooled PBS to resuspend the precipitate; 4°C, 100000-120000g ultra-high speed centrifugation for 60-120 minutes, discarding the supernatant, thereby obtaining the stem cell extracellular vesicles.

[0032] The second aspect of the present invention provides a pharmaceutical composition, which contains: (a) the stem cell extracellular vesicle preparation described in the first aspect of the present invention, and (b) a pharmaceutically acceptable carrier.

[0033] In another preferred embodiment, the pharmaceutical composition is a pharmaceutical composition free of cells and cell fragments.

[0034] In another preferred embodiment, the “cell-free” refers to that the pharmaceutical composition does not contain living cells and dead cells.

[0035] In another preferred embodiment, the pharmaceutical composition comprises bioactive factors related to anti-inflammation or tissue damage repair, and the bioactive factors include MMP1, HGF, VEGF or a combination thereof.

[0036] In another preferred embodiment, the pharmaceutically acceptable carrier is selected from the following group: sodium chloride, sodium phosphate, polyethylene glycol, chitosan, sodium hyaluronate, trehalose, heparin, or a combination thereof.

[0037] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the following group: liquid dosage form, solid dosage form (such as lyophilized dosage form).

[0038] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the following group: aerosol inhalation, eye drops, nasal drops, injection.

[0039] In another preferred embodiment, the pharmaceutical composition is an aerosol inhaler.

[0040] In another preferred embodiment, the pharmaceutical composition has the following characteristics: its main active ingredient is easily taken up by the tiny bronchial epithelium, alveolar epithelial cells, and inflammatory cells in the airways, thereby producing significant effects of inhibiting airway inflammatory response, repairing airway damage, and improving airway tension, and can be used to prevent and treat respiratory inflammatory diseases and injuries caused by various reasons.

[0041] In another preferred embodiment, the pharmaceutical composition is used to treat acute respiratory inflammation and / or chronic respiratory inflammation.

[0042] In another preferred embodiment, the pharmaceutical composition is used to treat chronic respiratory inflammation, preferably for treating chronic respiratory inflammation characterized by infiltration of inflammatory cells such as eosinophils, neutrophils, monocytes and macrophages in the respiratory mucosa, and more preferably for treating bronchial asthma and chronic obstructive pulmonary disease (COPD).

[0043] The third aspect of the present invention provides a method for preparing a pharmaceutical composition containing stem cell extracellular vesicles, the method comprising the steps of:

[0044] (S1) culturing human stem cells (such as adipose-derived mesenchymal stem (progenitor) cells) to a predetermined confluence (such as 75-90%);

[0045] (S2) continuing to culture the cells for a period of time T1 under conditions suitable for producing EVs; wherein T1 is generally 24-72 hours, preferably 30-60 hours;

[0046] (S3) removing cells from the culture system, thereby isolating and obtaining a culture medium containing stem cell extracellular vesicles, which is a "conditioned medium";

[0047] (S4) mixing the conditioned medium with polyethylene glycol (PEG) to form a first mixture, and placing the mixture for a period of time T2 to form PEG-modified stem cell extracellular vesicles; wherein the T2 is generally 6-60 hours, preferably 12-48 hours;

[0048] (S5) centrifuging the first mixture in the previous step to precipitate the PEG-modified stem cell extracellular vesicles, and discarding the supernatant to obtain a PEG-modified stem cell extracellular vesicle precipitate;

[0049] (S6) resuspending the PEG-modified stem cell extracellular vesicle precipitate obtained in the previous step to obtain a first resuspended mixture;

[0050] (S7) centrifuging the first resuspended mixture to precipitate the PEG-modified stem cell extracellular vesicles, and discarding the supernatant to obtain a PEG-modified stem cell extracellular vesicle precipitate;

[0051] (S8) Resuspending the PEG-modified stem cell extracellular vesicle precipitate obtained in the previous step to obtain a medically usable stem cell extracellular vesicle preparation.

[0052] In another preferred embodiment, in the stem cell extracellular vesicle preparation, the content of MMP-1 derived from extracellular vesicles is 1500-2000 pg / ml, and the content of HGF is 5500-6000 pg / ml.

[0053] In another preferred embodiment, the method further comprises:

[0054] (S9) Mixing the medically acceptable stem cell extracellular vesicle preparation (or active substance) with a pharmaceutically acceptable carrier to prepare a pharmaceutical composition.

[0055] In another preferred embodiment, the method further comprises preparing the pharmaceutical composition into an aerosol inhalation preparation, an injection, or a lyophilized preparation.

[0056] The fourth aspect of the present invention provides a use of the stem cell extracellular vesicle preparation described in the first aspect of the present invention or the pharmaceutical composition described in the second aspect of the present invention for preparing a drug or preparation for treating respiratory inflammation.

[0057] In another preferred embodiment, the respiratory tract inflammation includes acute respiratory tract inflammation and chronic respiratory tract inflammation.

[0058] In another preferred embodiment, the chronic respiratory inflammation is mainly characterized by the infiltration of inflammatory cells such as eosinophils, neutrophils, monocytes and macrophages in the respiratory mucosa.

[0059] In another preferred embodiment, the chronic respiratory inflammation is bronchial asthma, chronic obstructive pulmonary disease (COPD), or a combination thereof.

[0060] In another preferred embodiment, the acute respiratory tract inflammation includes viral infectious inflammation, bacterial infectious inflammation, fungal infectious inflammation, or a combination thereof.

[0061] In another preferred embodiment, the acute respiratory inflammation includes a disease selected from the following group: acute respiratory distress syndrome (ARDS), acute lung injury (ALI), novel coronavirus (SARS-CoV-2) infectious pneumonia, or a combination thereof.

[0062] The fifth aspect of the present invention provides a method for treating respiratory inflammation, comprising the steps of: administering the pharmaceutical composition of the second aspect of the present invention to a subject in need thereof.

[0063] In another preferred embodiment, the respiratory tract inflammation includes acute respiratory tract inflammation and chronic respiratory tract inflammation.

[0064] In another preferred embodiment, the chronic respiratory inflammation is mainly characterized by the infiltration of inflammatory cells such as eosinophils, neutrophils, monocytes and macrophages in the respiratory mucosa.

[0065] In another preferred embodiment, the chronic respiratory inflammation is bronchial asthma, chronic obstructive pulmonary disease (COPD), or a combination thereof.

[0066] In another preferred embodiment, the acute respiratory tract inflammation includes viral infectious inflammation, bacterial infectious inflammation, fungal infectious inflammation, or a combination thereof.

[0067] In another preferred embodiment, the acute respiratory inflammation includes a disease selected from the following group: acute respiratory distress syndrome (ARDS), acute lung injury (ALI), novel coronavirus (SARS-CoV-2) infectious pneumonia, or a combination thereof.

[0068] In another preferred embodiment, the subject in need is a human.

[0069] In another preferred embodiment, the subject in need suffers from acute respiratory inflammation and / or chronic respiratory inflammation.

[0070] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1The biological characteristics of the main active ingredients in the pharmaceutical composition preparation of human adipose-derived mesenchymal stem cell extracellular vesicles are shown.

[0072] Figure 2 The in vitro cellular uptake of the main active ingredients in the pharmaceutical composition preparation of human adipose-derived mesenchymal stem cell extracellular vesicles is shown.

[0073] Figure 3 The biodistribution of DiR-labeled human adipose-derived mesenchymal progenitor cell exosomes in mice is shown. (A) The distribution of exosomes in mice 1h, 4h, 24h, 48h and 72h after aerosol administration of exosomes; (B) The distribution of exosomes in mouse ex vivo tissues 48h and 72h after aerosol administration of exosomes.

[0074] Figure 4 The figure shows the effect of human adipose-derived mesenchymal progenitor cell exosome aerosolization on PA-induced pneumonia in mice. (A) Morphological observation of mouse lung tissue HE staining; (B) Mouse lung tissue injury score; (C) and (D) Changes in the number of total leukocytes and neutrophils in mouse bronchoalveolar lavage fluid; (E) and (F) Pro-inflammatory cytokines TNF-α and IL-6 in mouse bronchoalveolar lavage fluid; (G) Anti-inflammatory cytokine IL-10 content in mouse bronchoalveolar lavage fluid. *P<0.05, **P<0.01, ****P<0.0001.

[0075] Figure 5 The changes in chest CT of patients with severe COVID-19 before and after aerosol treatment of exosomes from human adipose-derived mesenchymal progenitor cells are shown. (A and B) Chest CT images of patients A and B before and after treatment, showing that the white flake shadows in their lungs were weakened after treatment.

[0076] Figure 6 The top 20 miRNAs and their relative expression levels in exosomes of human adipose-derived mesenchymal progenitor cells produced in three batches are shown.

[0077] Figure 7 Schematic diagram of the preparation of an asthma mouse model showing mixed granulocyte characteristics.

[0078] Figure 8 Schematic diagram showing the pharmaceutical composition preparation of human adipose-derived mesenchymal stem cell extracellular vesicles for treating asthmatic mice with mixed granulocytic characteristics.

[0079] Fig. 9 A schematic diagram showing the treatment of COPD mice with a pharmaceutical composition preparation of human adipose-derived mesenchymal stem cell extracellular vesicles. DETAILED DESCRIPTION

[0080] After extensive and in-depth research, the inventors unexpectedly developed a cell-free biopharmaceutical preparation that can effectively treat chronic respiratory inflammation for the first time. The pharmaceutical preparation contains stem cell extracellular vesicles derived from human stem cells, has a specific particle size, and contains biologically active factors related to anti-inflammatory or tissue damage repair. The stem cell vesicles of the present invention are prepared by polyethylene glycol (PEG) precipitation method. The particle size of the extracellular vesicles is concentrated between 80-120nm, the particle size is smaller and uniform, and has extremely excellent dispersibility in medical solvents (such as saline, etc.), and is particularly suitable for direct application to the patient's respiratory tract by atomization inhalation and other methods; and the main active ingredients in the pharmaceutical composition or preparation of the stem cell extracellular vesicles of the present invention are easily taken up by fine bronchial epithelial cells, alveolar epithelial cells, and inflammatory cells in the airway, thereby quickly and efficiently treating or alleviating chronic respiratory inflammation, especially diseases such as asthma and COPD. On this basis, the present invention is completed.

[0081] the term

[0082] asthma

[0083] Asthma generally refers to bronchial asthma, a heterogeneous disease characterized by chronic inflammation of the airways involving multiple cells (such as eosinophils, mast cells, T lymphocytes, neutrophils, airway epithelial cells, etc.) and cell components. This chronic inflammation is associated with airway hyperresponsiveness, and usually presents with extensive and variable reversible expiratory airflow limitation, leading to recurrent symptoms of wheezing, shortness of breath, chest tightness, and (or) coughing, with intensity varying over time. Most of the symptoms occur and intensify at night and (or) early morning, and most patients can relieve themselves or with treatment. If bronchial asthma is not diagnosed and treated in time, irreversible airway narrowing and airway remodeling may occur as the course of the disease prolongs.

[0084] Chronic obstructive pulmonary disease (COPD)

[0085] Chronic obstructive pulmonary disease (COPD) is a common preventable and treatable disease characterized by persistent airflow limitation. The airflow limitation progresses and is related to the enhanced chronic inflammatory response of the airways and lungs to toxic particles or gases. It is a common chronic disease characterized by chronic bronchitis and / or emphysema with airflow obstruction, which can further develop into cor pulmonale and respiratory failure.

[0086] Mesenchymal stem cell extracellular vesicles

[0087] There are three main types of stem cell extracellular vesicles (EVs): exosomes, microvesicles, and apoptotic bodies. All three main types of EVs are encapsulated by a lipid bilayer and have a diameter ranging from 30 to 2000 nm.

[0088] The term exosomes refers to a subset of EVs with a diameter of 50–100 nm that are derived from endosomes and are the major components of paracrine secretions of various cell types, including mesenchymal stem cells (MSCs).

[0089] MSCs exosomes are a type of MSCs-derived EVs with a diameter ranging from 50-100 nm and a complete lipid bilayer membrane structure.

[0090] Exosomes are a type of cargo-rich carrier that functions mainly through the continuous transport of microRNAs (miRNAs) and proteins. More than 150 miRNAs and more than 850 unique proteins have been identified in MSCs-derived exosomes, which alter various activities of target cells through different pathways. MSC exosomes are involved in physiological and pathological processes such as body development, epigenetic regulation, immune regulation (miR-155 and miR-146), tumorigenesis and tumor progression (miR-23b, miR-451, miR-223, miR-24, miR-125b, miR-31, miR-214 and miR-122). According to ExoCarta, more than 900 proteins have been collected from MSCs exosomes. Some studies have shown that MSC exosomes carry some cytokines and growth factors, such as TGFβ1, interleukin-6 (IL-6), IL-10 and hepatocyte growth factor (HGF), which have been shown to contribute to immune regulation. Vascular endothelial growth factor (VEGF), extracellular matrix metalloproteinase inducer (EMMPRIN) and MMP-9 have all been reported in MSC exosomes. These three proteins play an important role in stimulating angiogenesis and may be the basis for the tissue repair effect of exosomes.

[0091] Preparation method of stem cell extracellular vesicles and pharmaceutical composition containing same of the present invention

[0092] The present invention provides an extracellular vesicle derived from stem cells, wherein the particle size of the extracellular vesicle is 30-200 nm, preferably 50-150 nm, and more preferably 80-120 nm; and the extracellular vesicle contains biologically active factors related to anti-inflammatory or tissue damage repair, wherein the active factors include matrix protein metalloenzyme 1 (MMP-1) and hepatocyte growth factor (HGF), wherein the content of MMP-1 is 1500-2000 pg / ml, and the content of HGF is 5500-6000 pg / ml.

[0093] The stem cells include, but are not limited to, stem cells derived from human umbilical cord blood, stem cells derived from human peripheral blood, human umbilical cord mesenchymal stem cells, human placental mesenchymal stem cells, human adipose mesenchymal stem cells, and human bone marrow-derived stem cells. In a preferred embodiment of the present invention, the stem cells are human adipose mesenchymal stem cells.

[0094] The present invention provides a method for preparing a pharmaceutical composition or preparation with key active substances in stem cell extracellular vesicles as main effective ingredients, and the specific technical scheme is as follows:

[0095] In a preferred embodiment, GMP-grade human adipose-derived mesenchymal stem cells are mainly used as parent cells for producing stem cell extracellular vesicles, but stem cells derived from tissues such as umbilical cord blood, umbilical cord, placenta, and bone marrow may also be used.

[0096] The preparation of the stem cell extracellular vesicle pharmaceutical composition of the present invention comprises the following steps:

[0097] 1) collecting the culture supernatant of the above-mentioned GMP-scaled human adipose-derived mesenchymal stem cells that have been effectively conditioned, performing differential centrifugation and filtration to obtain a conditioned medium, and then performing vesicle separation in combination with a polymer precipitation method, wherein the polymer includes but is not limited to polyethylene glycol (PEG3000-9000) of different molecular weights and compositions of different ratios (1:1-1:6);

[0098] 2) Prepare a PEG mother solution of appropriate concentration (8% to 30% g / ml) with PBS and filter it, mix it with the above-treated conditioned medium in a 1:1 volume ratio, and incubate it overnight at low temperature (4 to 8°C);

[0099] 3) After centrifugation (4°C, 3000-5000g, 45-60min), discard the supernatant, add pre-cooled PBS to resuspend the precipitate, and discard the supernatant after ultra-high-speed centrifugation (4°C, 100000-120000g, 1-2h);

[0100] 4) Add an appropriate volume (3-10 ml) of medical saline to resuspend the precipitate, and then identify the biological characteristics of the extracellular vesicles of human adipose-derived mesenchymal stem cells by particle size analysis, scanning electron microscopy observation, and immunoblotting;

[0101] 5) Detecting the key active substance indicators of the main effective components in the extracellular vesicles of the stem cells;

[0102] 6) Using 3-10 ml of a pharmaceutically acceptable carrier or excipient to dilute the stem cell extracellular vesicles obtained above to an appropriate effective concentration.

[0103] According to an embodiment of the present invention, the preparation of the pharmaceutical composition of stem cell extracellular vesicles is preferably allogeneic human adipose-derived mesenchymal stem cell extracellular vesicles.

[0104] According to an embodiment of the present invention, the pharmaceutical composition or preparation of stem cell extracellular vesicles contains pharmaceutically acceptable carriers or excipients, including but not limited to sodium chloride, sodium phosphate, polyethylene glycol, chitosan, sodium hyaluronate, trehalose, heparin, etc., and any combination of the above substances.

[0105] In addition, the pharmaceutical composition or preparation of human adipose-derived mesenchymal stem cell extracellular vesicles produced was used for in vitro cell uptake studies, including type II alveolar epithelial cells A549 and activated macrophages RAW247.6. The active ingredient of the pharmaceutical composition of human adipose-derived mesenchymal stem cell extracellular vesicles labeled with lipophilic fluorescent dye PKH67 (1×10 7 ~4×10 8 After adding the particles to the cells and culturing for 24 to 48 hours, the active ingredients of the composition were observed under an inverted fluorescence microscope. The results showed that the active ingredients of the composition could be taken up by the cells.

[0106] Scale-up of the process for producing stem cell extracellular vesicles under GMP conditions

[0107] One HyperFlask cell factory can produce 500 ml of conditioned medium containing stem cell extracellular vesicles, which is expected to separate 5×10 10 -10×10 10 Total amount of extracellular vesicles in stem cells.

[0108] At present, one production unit can separate stem cell extracellular vesicles from two cell factories with a total of 800-1200 ml of conditioned medium, and it is expected to separate 2-5×10 11 Stem cell extracellular vesicles, 2-5×10 per patient 9 One production unit can produce enough drugs for 100 to 250 patients in one batch.

[0109] Application of stem cell extracellular vesicles and pharmaceutical compositions containing same

[0110] The present invention also provides the use of a pharmaceutical composition or preparation containing key active substances in stem cell extracellular vesicles as main effective ingredients for treating respiratory inflammatory diseases.

[0111] The pharmaceutical composition of the present invention contains (a) the stem cell extracellular vesicle preparation according to the first aspect of the present invention, and (b) a pharmaceutically acceptable carrier.

[0112] In another preferred embodiment, the pharmaceutical composition is a pharmaceutical composition free of cells and cell fragments.

[0113] In the present invention, preferably, the active ingredients also include bioactive factors related to anti-inflammatory or tissue damage repair, including but not limited to MMP1, HGF, or VEGF. These bioactive factors are mainly present in stem cell extracellular vesicles. In addition, some bioactive factors may exist outside stem cell extracellular vesicles.

[0114] In the present invention, a particularly preferred pharmaceutical preparation is an aerosol inhalation preparation.

[0115] In a preferred embodiment, a mouse asthma model characterized by mixed granulocytes is used to verify the application of the pharmaceutical composition of the present invention in treating respiratory inflammatory diseases.

[0116] First, a pharmaceutical composition (1×10 7 ~1×10 8 The distribution of the drug composition in the lungs of mice with asthma was studied by inhaling the drug into the respiratory tract. It was found that the main active ingredients of the drug composition were easily taken up by the small bronchial epithelium, alveolar epithelial cells, and inflammatory cells in the airways. In addition, after the mice were randomly divided into groups (Table 1), the mixed granulocyte-characterized mouse asthma model was used. After treatment with dexamethasone, stem cells, and different doses of the main active ingredients of the drug composition, the results showed that the main active ingredients of the drug composition were significantly increased at low doses (1×10 5 ~1×10 8 particles / mouse) and high doses (2×10 6 ~2×10 9 When the asthma of mice was treated with the condition of 1×10 particles / mouse, the results were better than those of the conventional drug dexamethasone (1-2 mg / kg) and stem cells (1×10 4 ~1.5×10 5cells / time / mouse) treatment more effectively reduced airway hyperresponsiveness and inflammatory factor levels, thereby effectively improving lung tissue pathological characteristics.

[0117] Table 1 The drug composition of human adipose-derived mesenchymal stem cells extracellular vesicles in the treatment of mixed granulocytic asthma mice

[0118]

[0119] In addition, in a preferred embodiment, a nicotine (1.5-5 mg)-induced COPD mouse model was first established. The control group mice were treated with nebulized saline via airway inhalation, while the experimental group mice were treated with a pharmaceutical composition of human adipose-derived mesenchymal stem cell extracellular vesicles (1×10 5 ~1×10 8 The mice were treated with aerosol inhalation of 100 mg / kg of chloramphenicol (Citrus particles / mouse) through the airway, once a day in the aerosol box, for 5 consecutive days. The lung function indexes of the mice were tested and it was found that their lung function was improved.

[0120] The main advantages of the present invention include:

[0121] (1) The stem cell extracellular vesicles of the present invention are prepared by a polyethylene glycol (PEG) precipitation method, and the particle size of the extracellular vesicles is concentrated between 80-120 nm, and the particle size is smaller and more uniform.

[0122] (2) The stem cell extracellular vesicles of the present invention contain bioactive factors related to anti-inflammation or tissue damage repair, and the content is higher than that of the extracellular vesicles prepared by traditional centrifugation methods.

[0123] (3) The main active ingredients in the stem cell extracellular vesicle pharmaceutical composition or preparation of the present invention are easily taken up by the small bronchial epithelial cells, alveolar epithelial cells, and inflammatory cells in the airways.

[0124] (4) Compared with conventional drugs or stem cell treatments, the pharmaceutical composition or preparation of stem cell extracellular vesicles of the present invention can more effectively improve or treat respiratory inflammatory diseases.

[0125] The present invention will be further described below in conjunction with specific implementation. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0126] Experimental materials and instruments

[0127] In a preferred embodiment of the present invention, human adipose-derived mesenchymal stem cells are used as parent cells for obtaining extracellular vesicles. Other experimental materials and instruments involved mainly include:

[0128] Wild-type BALB / c mice (7 weeks old, female, SPF grade), wild-type C57BL / 6 mice (8-10 weeks old, male, SPF grade), anesthetics (50 mg / kg ketamine and 30 mg / kg xylazine), type II alveolar epithelial cells A549, RAW264.7 macrophages, multidrug-resistant aeruginosa (PA), chicken ovalbumin (OVA, Sigma-Aldrich), aluminum hydroxide, cigarette smoke, lipopolysaccharide (LPS), dexamethasone (Dex), methacholine, medical saline, PEG of different molecular weights, sterile phosphate buffer (PBS), 1 ml sterile syringe and 20G needle, PKH67 dye, DiR dye, paraffin block, H&E staining solution, PAS staining solution, 10% neutral formaldehyde fixative, OCT embedding medium, liquid nitrogen, Illumina TruSeq Small RNA kit, ELISA detection kits for mouse TNF-α, IgE, IL-4, IL-5, IL-6, IL-10, IL-13 and IL-17A, centrifuge tubes, 12-well cell culture plates, centrifuge, paraffin embedding machine, paraffin slicer, frozen slicer, ordinary bright field microscope (40× objective lens), upright fluorescence microscope (40× objective lens), inverted fluorescence microscope (20× objective lens), cell culture incubator, nebulizer box, non-invasive pulmonary function instrument, blood cell counter, IVIS imaging system, particle size analyzer, Illumina sequencing platform.

[0129] Data Statistical Analysis Methods

[0130] All experimental data were statistically analyzed using GraphPad Prism 6.0 software and expressed as mean ± standard error (Mean ± SEM). When comparing two groups of data, Mann-Whitney U test was used for data comparison analysis; when comparing multiple groups of data, One-way ANOVA and Tukey were used for data comparison analysis. P values ​​less than 0.05 were considered to be significantly different.

[0131] Example 1

[0132] Preparation of stem cell extracellular vesicles pharmaceutical composition

[0133] In all embodiments of the present invention, human adipose-derived mesenchymal stem cells with GMP grade are selected as parent cells for producing stem cell extracellular vesicles, but stem cells derived from tissues such as umbilical cord blood, umbilical cord, placenta, and bone marrow may also be used.

[0134] The main steps of preparing the stem cell extracellular vesicle preparation of the present invention are as follows:

[0135] (1) collecting the culture supernatant of the above-mentioned GMP-scaled human adipose-derived mesenchymal stem cells that have been effectively conditioned, and removing cell debris by differential centrifugation and filtration to obtain a conditioned medium containing extracellular vesicles;

[0136] (2) In this step, separation is performed by polymer precipitation method, preferably PEG9000; 20% g / ml PEG9000 mother solution is prepared with PBS, filtered with a 0.22 μm filter, and then added to the conditioned medium treated in the previous step at a volume ratio of 1:1, incubated at 4°C overnight, and then centrifuged at 4°C, 4000g for 50 min, the supernatant is discarded and the precipitate is retained;

[0137] (3) Add pre-cooled PBS to resuspend the precipitate, centrifuge at 120,000 g for 1 h at 4°C, remove the supernatant, add 5 ml of medical carrier solution to resuspend the precipitate, and obtain a sample without PEG precipitate, which is used as the test group sample;

[0138] (4) The samples in the experimental group were further subjected to particle size analysis, morphological observation by scanning electron microscopy, and immunoblotting identification of marker proteins such as CD9, CD63, CD81, and TSG101 to confirm that the extracellular vesicles of human adipose-derived mesenchymal stem cells ( Figure 1 );

[0139] (5) Detect the key active substances of the main effective ingredients in the vesicles and verify their pharmacological potential in vitro;

[0140] (6) Diluting the stem cell extracellular vesicle preparation obtained above to an appropriate effective concentration using a medical carrier solution, thereby obtaining a stem cell extracellular vesicle pharmaceutical composition.

[0141] At the same time, a control group was set up to prepare stem cell extracellular vesicle preparations, and the preparation method was as follows:

[0142] (1) collecting the culture supernatant of the above-mentioned GMP-scaled human adipose-derived mesenchymal stem cells that have been effectively conditioned, and removing cell debris by differential centrifugation and filtration to obtain a conditioned medium containing extracellular vesicles;

[0143] (2) The conditioned medium containing extracellular vesicles treated in the previous step was subjected to ultrahigh-speed centrifugation at 4°C and 120,000 g for 1 h, the supernatant was removed, and 5 ml of the medical carrier solution was added to resuspend the precipitate to obtain a control group sample.

[0144] The results showed that compared with the control group, the method of the present invention can obtain more stem cell extracellular vesicle preparations with uniformly dispersed particles and transparency, and the drug preparation is rich in bioactive factors related to anti-inflammation and tissue damage repair, such as MMP-1 and HGF (Table 2).

[0145] Table 2. Stem cell extracellular vesicles are rich in bioactive factors related to anti-inflammation and tissue damage repair

[0146]

[0147] Among them, the content of MMP-1 (with anti-fibrosis effect) is at least 200 times higher than that of the control group, and the content of HGF (with certain anti-inflammatory effect) is about 2500 times higher. In addition, factors such as VEGF also increase by a corresponding amount. This shows that the stem cell extracellular vesicle preparation prepared by the present invention has excellent anti-inflammatory and anti-fibrosis properties.

[0148] The stem cell extracellular vesicle pharmaceutical composition (500 mL) prepared by the present invention contains 1.7×10 11 The particle size of the extracellular vesicles is 30-200 nm, preferably 50-150 nm, and more preferably 80-120 nm, which is more conducive to the therapeutic effect after administration.

[0149] In addition, no PEG component was detected in the stem cell extracellular vesicle pharmaceutical composition in the experimental group (below the detection limit).

[0150] Example 2

[0151] In vitro cellular uptake of stem cell extracellular vesicle pharmaceutical compositions

[0152] In order to detect the cellular uptake of the main active ingredients of the stem cell extracellular vesicles pharmaceutical composition, the active ingredients of the human adipose-derived mesenchymal stem cell extracellular vesicles pharmaceutical composition were first labeled with the lipophilic fluorescent dye PKH67 and added to type II alveolar epithelial cells A549 and LPS-stimulated RAW264.7 macrophages, respectively. After culturing for 24 hours and 48 hours, the cellular uptake of the active ingredients in the composition was observed under an inverted fluorescence microscope. The main steps of the implementation are as follows:

[0153] (1) The main active ingredient (2×10 7 extracellular vesicles);

[0154] (2) Type II alveolar epithelial cells A549 and RAW264.7 macrophages were cultured at 1×10 4 Cells / cm2 and 5×10 4 Cells / cm 2 The cells were seeded at different densities in 12-well culture plates and cultured in a cell culture incubator (37°C, 5% CO2) for 24 h;

[0155] (3) adding the labeled pharmaceutical composition to a type II alveolar epithelial cell A549 culture plate, culturing for 24 hours, and observing the uptake of the active ingredient in the pharmaceutical composition by the cells under an inverted fluorescence microscope;

[0156] (4) RAW264.7 macrophages were stimulated with 10 ng / ml LPS and then added with the labeled pharmaceutical composition. The cells were cultured for 48 h, and the culture dish was removed to observe the uptake of the active ingredient in the pharmaceutical composition by the cells under an inverted fluorescence microscope.

[0157] The results showed that the active ingredients of the drug composition of human adipose-derived mesenchymal stem cell extracellular vesicles could be taken up by type II alveolar epithelial cells A549 and significantly accumulated in the cells; at the same time, the active ingredients of the drug composition of stem cell extracellular vesicles could also be taken up by activated macrophages ( Figure 2 ).

[0158] Example 3

[0159] Study on the biodistribution of fluorescently labeled EVs by aerosol inhalation in mice

[0160] In this example, the distribution and duration of extracellular vesicles of human adipose-derived mesenchymal stem cells in mouse lung tissue after aerosol inhalation were studied. The fluorescent labeling extracellular vesicle tracing method was used to explore it.

[0161] Human adipose-derived mesenchymal stem cells extracellular vesicles (3×10 9 The DiR-labeled extracellular vesicles (EVs) were nebulized for 30 minutes and inhaled into the lungs of mice (N=3). The distribution of DiR-labeled EVs in the lung tissue of mice was observed by in vivo fluorescence imaging at 1h, 4h, 24h, 48h and 72h after nebulization inhalation, and their retention time in the lungs of mice was analyzed.

[0162] like Figure 3 As shown in A, the distribution of DiR-labeled human adipose-derived mesenchymal stem cell extracellular vesicles in the mouse body was observed from different angles at different time points after the atomization needle was used to atomize and inhale the extracellular vesicles into the mouse trachea. The results showed that ( Figure 3 A), in addition to the fluorescent signal of esophageal reflux caused by intratracheal administration ( Figure 3 A abdomen), the fluorescent signal in the mouse is mainly concentrated in the mouse lung tissue ( Figure 3A back, right side and left side), especially 4 hours after administration, the fluorescence signal in the left lung tissue of mice was the highest, while that in the right lung was weaker, indicating that a large number of extracellular vesicles accumulated in the left lung lobe at this time; then, the fluorescence signal in the left lung lobe decreased sharply, while that in the right lung lobe gradually weakened, indicating that the extracellular vesicles were gradually consumed and reduced in the lung tissue. Since the fluorescence signal in the tissue cannot be accurately observed in vivo, various tissues were taken for in vitro fluorescence imaging observation. Figure 3 As shown in B, compared with the control group, no fluorescence signals were found in the heart, liver, spleen, kidney and trachea 48h and 72h after intratracheal aerosol administration of extracellular vesicles, while fluorescence signals were only found in the lung tissue. The fluorescence signal was stronger at 48h and weaker at 72h.

[0163] In summary, the results show that after intratracheal aerosol administration, extracellular vesicles are mainly distributed in lung tissue, and are almost not distributed in other tissues.

[0164] Example 4

[0165] Study on the effectiveness of aerosol inhalation of EV in the treatment of infectious lung injury animal model

[0166] In this example, the effectiveness of human adipose-derived mesenchymal stem cell extracellular vesicles in treating multidrug-resistant aeruginosa (PA)-induced mouse pneumonia model after aerosol inhalation was studied. A blank control group of PBS-induced mice, a PA-induced mouse pneumonia group, a human adipose-derived mesenchymal stem cell extracellular vesicle aerosol inhalation treatment of PA-induced mouse pneumonia group, and a mouse fibroblast extracellular vesicle treatment of mouse pneumonia group were set up. Each group consisted of 6 8-10 week-old C57BL / 6 wild-type male mice, and the dosage of extracellular vesicles for each mouse in the treatment group was 1×10 6 The extracellular vesicles / g of stem cells were calculated. After 4 hours of induction of multidrug-resistant aeruginosa, extracellular vesicles were aerosolized and inhaled once a day for 5 days. Afterwards, bronchoalveolar lavage fluid was collected for neutrophil count and related inflammatory factor levels. In addition, morphological observation of lung tissues and lung injury assessment were performed by HE staining.

[0167] The results are as follows Figure 4 As shown in the figure, morphological observation of lung tissue HE staining showed that compared with the control group, the lung injury of mice with PA-induced pneumonia treated with human adipose-derived mesenchymal stem cell extracellular vesicles aerosol was significantly improved ( Figure 4 A), and the lung tissue scoring results also showed that the lung injury score of the mice was significantly reduced, while the lung injury score of the mice treated with mouse fibroblast extracellular vesicles was not significantly reduced ( Figure 4B). In addition, the results of the total white blood cell and neutrophil counts in the bronchoalveolar lavage fluid of mice revealed that the number of total white blood cells and neutrophil counts in the bronchoalveolar lavage fluid of mice treated with human adipose-derived mesenchymal stem cell extracellular vesicle aerosol was significantly reduced compared with the control group ( Figure 4 C, D); the levels of pro-inflammatory cytokines TNF-α and IL-6 in their bronchoalveolar lavage fluid were also significantly reduced ( Figure 4 E, F), while the level of anti-inflammatory cytokine IL-10 was significantly increased ( Figure 4 G).

[0168] In summary, these results indicate that human adipose-derived mesenchymal stem cell extracellular vesicle aerosol therapy can significantly inhibit PA-induced lung injury in mice and reduce the level of inflammatory response, thereby effectively treating this model of pneumonia in mice.

[0169] Example 5

[0170] Clinical trial of nebulized EV inhalation for the treatment of acute lung injury caused by COVID-19

[0171] In this example, the safety and efficacy of allogeneic adipose-derived mesenchymal stem cell extracellular vesicles inhaled by aerosol inhalation in the treatment of severe and critical patients with novel coronavirus pneumonia (COVID-19) were preliminarily explored.

[0172] It is planned to enroll 30 severe and critical NCP subjects and divide them into three dose groups: low, medium and high. In addition to routine ICU treatment, all subjects will be given aerosol inhalation of allogeneic fat MSCs-Exo treatment, 2-8×10 8 Stem cell extracellular vesicles / 3ml, once a day for 5 consecutive days. The incidence of adverse reactions and clinical improvement time within 28 days, as well as the number of patients weaned off the machine, the number of ICU nursing days, the duration of mechanical ventilation for survivors and the mortality rate were recorded and analyzed. In addition, the improvement of patients' symptoms after treatment was judged by daily SOFA scores, detection of relevant biochemical indicators and lung imaging observations, and the time when the new coronavirus turned negative in the patient's respiratory specimens. The inclusion criteria of this study mainly include 1) age 18-75 years old, regardless of gender, and the individual or family members voluntarily joined and signed the informed consent form; 2) RT-PCR test was positive or clearly diagnosed as a patient with new coronavirus pneumonia; 3) met the diagnostic criteria for severe and critical patients. The exclusion criteria mainly include 1) patients who are carriers of related viruses or have severe allergic constitutions, and patients with pneumonia caused by other viruses; 2) patients with lung cancer or long-term use of immunosuppressive drugs; 3) patients undergoing hemodialysis or peritoneal dialysis, and patients with abnormal liver function; 4) patients who are using ECMO or high-frequency oscillation ventilation; 5) patients who are pregnant, breastfeeding, or planning to conceive within six months; 6) patients who are judged by the researchers to be unable to participate in the study or who fail to understand and implement the plan.

[0173] In the completed exploratory clinical study of human adipose-derived mesenchymal stem cell extracellular vesicles (haMSC-Exos) for the treatment of severe novel coronavirus pneumonia (COVID-19), in addition to verifying its safety in patients with severe respiratory diseases, it was also observed that after hMSC-Exos aerosol treatment, the chest imaging of COVID-19 patients showed significant improvement ( Figure 5 A, B), the subjects' lung exudation improved compared with before. At the same time, there was a downgrade in respiratory support. In addition, 2 cases were weaned from ventilator oxygen therapy on the 4th and 5th days after the first treatment. Within 7-14 days after the first treatment, the clinical symptoms of all subjects improved, and the clinical symptom improvement rate was 100%. They met the discharge criteria and were discharged. Within 14-28 days after the first treatment, the clinical symptoms of 6 subjects continued to improve, and the improvement rate was 85.7%.

[0174] In summary, the results of this clinical trial show that aerosolization of extracellular vesicles of human adipose-derived mesenchymal stem cells can assist in the treatment of acute lung injury induced by the new coronavirus to a certain extent, thereby improving the pneumonia symptoms of critically ill patients, and it is safe.

[0175] Example 6

[0176] Extracellular vesicles of human adipose-derived mesenchymal stem cells are rich in anti-inflammatory related miRNAs

[0177] In this embodiment, the expression of microRNAs (miRNAs) in three batches of human adipose-derived mesenchymal stem cell extracellular vesicles (haMSC-Exos) was studied. Three batches of human adipose-derived mesenchymal stem cell extracellular vesicle products produced under GMP conditions were selected for total microRNA extraction. The library was constructed using the Illumina TruSeq Small RNA kit, including connecting the 3' end adapter and the 5' end adapter respectively, and the random primers were reversed into the first chain cDNA, followed by library enrichment and gel purification. After the library was quantified, it was sampled and tested in proportion, and the Illumina platform was used for sequencing. Finally, the quality-controlled data, i.e., clean data (reads), was compared with the human genome data, the expression levels of known miRNAs in each sample were statistically analyzed, and the expression levels were normalized using TPM (Transcripts Per Million).

[0178] The results showed that a total of 400 known miRNAs were identified in this sequencing. Among them, the statistics of the top 20 miRNAs and their relative expression levels are as follows: Figure 6As shown in the figure. Among these 20 known miRNAs, those with anti-inflammatory and immunomodulatory functions include hsa-let-7b-5p, hsa-let-7a-5p, hsa-miR-126-5p, hsa-miR-151a-3p, hsa-miR-1246, hsa-miR-21-5p, hsa-miR-146a-5p and hsa-let-7b-3p, etc., and thus can inhibit or weaken acute lung injury or ARDS. Bioinformatics prediction analysis showed that the main mechanism of these miRNAs to exert anti-inflammatory effects is dependent on targeted inhibition of TLR4 / MyD88 / NF-κB and / or JAK2 / STAT3 signaling pathway activation.

[0179] In summary, these results indicate that human adipose-derived mesenchymal stem cell extracellular vesicles can produce major biological functions depending on the miRNAs they contain, thereby exerting pharmacological effects in inflammatory diseases or injuries.

[0180] Example 7

[0181] The drug composition of stem cell extracellular vesicles can reach the site of lung airway inflammation through respiratory tract aerosol inhalation

[0182] In order to verify that the drug composition of stem cell extracellular vesicles can be more effectively distributed to the site of pulmonary airway inflammation after aerosol inhalation through the respiratory tract, its biodistribution in lung tissue was studied.

[0183] First, an asthma mouse model with a mixture of eosinophils and neutrophils as the main feature was established ( Figure 7 ). Seven-week-old female wild-type BALB / c mice were placed in an independent ventilation system in an SPF animal room for adaptive feeding for one week. Then, when the modeling group (OVA+LPS) mice were treated, a mixture of 10 μg of chicken ovalbumin (OVA) and 0.4 mg of aluminum hydroxide dissolved in 200 μl of normal saline was intraperitoneally injected into the mice for sensitization treatment on the same day and the 7th day; subsequently, the mice were challenged on the 14th, 15th, 16th and 17th days, and the mice were treated with a 6% OVA solution dissolved in medical normal saline for 25 minutes.

[0184] The experiment was divided into two groups: a control group (100 μl) of medical saline and a group of human adipose-derived mesenchymal stem cell extracellular vesicles labeled with the lipophilic fluorescent dye PKH67 (100 μl, 1×10 7The two groups of mice were treated with aerosol inhalation of extracellular vesicles (EVs) through the respiratory tract, and each group had 3 mice. After anesthesia, the two groups of mice were treated with aerosol inhalation on the 13th and 14th days, respectively. Then, 0.5h, 1h, 3h, 24h and 48h after administration were selected for IVIS fluorescence imaging and fluorescence intensity analysis of lung tissue in vitro. After that, the lung tissue was quickly frozen in liquid nitrogen, embedded in OCT, and frozen in liquid nitrogen again; a 10μm frozen section was prepared by a cryostat, dried at room temperature, and the OCT embedding agent was washed off, the cell nucleus was stained with DAPI, and the slide was sealed with a fluorescence quencher; then, a fluorescence microscope was used to image it and analyze the localization of PKH67-labeled EVs in the airways of lung tissue.

[0185] The results showed that the labeled extracellular vesicles were localized around the bronchi and blood vessels in the inflammatory areas of mouse lung tissue.

[0186] Example 8

[0187] Pharmaceutical composition of stem cell extracellular vesicles can reduce airway hyperresponsiveness in asthmatic mice

[0188] The degree of airway hyperresponsiveness depends mainly on the level of airway inflammation and individual immune response. It is an important feature of the presence of granulocyte inflammation in the asthma phenotype of children and adults. In order to explore whether the pharmaceutical composition of stem cell extracellular vesicles can improve granulocyte-mediated asthma by respiratory aerosol inhalation, the asthma model in Example 7 was used for treatment research ( Figure 8 ). The specific implementation method is as follows:

[0189] Seven-week-old female wild-type BALB / c mice were placed in an independent ventilation system in an SPF animal room for one week of adaptive feeding and then randomly divided into 6 groups, with 10 mice in each group. When the saline group (saline) and the modeling group (OVA+LPS) mice were treated, medical saline or a mixture of 10 μg chicken ovalbumin (OVA) and 0.4 mg aluminum hydroxide dissolved in 200 μl saline was injected intraperitoneally on the same day and the seventh day for sensitization treatment; subsequently, the mice were challenged on the 14th, 15th, 16th and 17th days, and the mice were treated with medical saline and its dissolved 6% OVA solution by atomization for 25 minutes, respectively, and 1 μg LPS was administered intratracheally only once on the 15th day. Among them, the mice in the drug treatment group were treated with medical saline, dexamethasone (1 mg / kg), adipose-derived mesenchymal stem cells (1.5×10 5 cells / times / mouse) and its vesicle pharmaceutical composition preparation (low-dose group 1×10 5 Extracellular vesicles / mouse, high-dose group 2×10 6The mice were treated with 100% extracellular vesicles / mouse) through the respiratory tract. 24 hours after the last challenge treatment (day 18), all mice were tested.

[0190] In order to detect the airway hyperresponsiveness of each group of mice, the mice were placed on a non-invasive pulmonary function tester and adapted for 10 minutes. Under the condition of stable breathing, the 5-minute airway resistance (RI value) of mice not treated with methacholine (Mch) was recorded. Then, the concentration of methacholine inhaled by aerosol was continuously increased (5, 10, 25 and 50 mg / ml), and the RI values ​​at the corresponding concentrations were detected respectively. Finally, the percentage curve of RI values ​​at different Mch doses was drawn.

[0191] The results showed that compared with dexamethasone and adipose-derived mesenchymal stem cell treatment, adipose-derived mesenchymal stem cell extracellular vesicles pharmaceutical composition preparation inhaled via respiratory tract aerosol can reduce mixed granulocyte-mediated airway hyperresponsiveness in asthmatic mice.

[0192] Example 9

[0193] Pharmaceutical composition preparation of stem cell extracellular vesicles can improve the pathological characteristics of lung tissue in asthmatic mice

[0194] In order to explore the pathological characteristics of lung tissue in asthmatic mice after respiratory aerosol inhalation of the stem cell extracellular vesicle pharmaceutical composition, the asthma animal model in Example 7 was used for research, and the specific operation was as follows:

[0195] (1) The lung tissue dry weight to wet weight ratio was used to evaluate the pulmonary edema in mice. The whole lung tissue after bronchoalveolar lavage fluid (BALF) was placed on gauze, the surface moisture was absorbed, and the wet weight was weighed and recorded; then it was placed in an 80°C incubator for 48 hours, weighed again, and the dry weight of the lung was recorded. Compared with dexamethasone and adipose-derived mesenchymal stem cell treatment, the drug composition preparation of adipose-derived mesenchymal stem cell extracellular vesicles can improve the pulmonary edema of mixed granulocyte-mediated asthmatic mice through respiratory aerosol inhalation.

[0196] (2) 5 ml of PBS was injected into the right ventricle to perfuse the mouse lung tissue, and 10% neutral formaldehyde solution was used for fixation for paraffin embedding and 4 μm slice preparation. Then, the slices were stained with H&E, and the field of view was randomly selected under a 40× objective lens for photography. Blind review scores were made based on the infiltration of inflammatory cells around the bronchi and blood vessels stained with H&E: no cell infiltration, recorded as 0; only some cells, recorded as 1, infiltrating cells formed a ring with a width of 1 cell, recorded as 2; infiltrating cells formed a ring with a width of 2 to 4 cells, recorded as 3; infiltrating cells formed a ring with a width of more than 4 cells, recorded as 4. The expected result is that compared with dexamethasone and adipose mesenchymal stem cell treatment, the pharmaceutical composition preparation of adipose mesenchymal stem cell extracellular vesicles can reduce the level of inflammatory cell infiltration around the lung bronchi and blood vessels of mixed granulocyte-mediated asthma mice through respiratory aerosol inhalation.

[0197] (3) The sections were stained with PAS, and the fields of view were randomly selected and photographed under a 40× objective. The number of goblet cells secreting mucus was scored blindly to evaluate the mucus secretion in the bronchi. No goblet cells were scored as 0; less than 25% goblet cells were scored as 1; 25% to 50% goblet cells were scored as 2; 50% to 75% goblet cells were scored as 3; more than 75% goblet cells were scored as 4.

[0198] The results showed that compared with dexamethasone and adipose-derived mesenchymal stem cell treatment, the drug composition preparation of adipose-derived mesenchymal stem cell extracellular vesicles can significantly reduce the level of mucus secretion of bronchial goblet cells in mixed granulocyte-mediated asthmatic mice through respiratory tract aerosol inhalation.

[0199] Example 10

[0200] The pharmaceutical composition of stem cell extracellular vesicles can reduce the level of inflammatory factors in asthmatic mice

[0201] In order to explore the levels of inflammatory factors in asthmatic mice after respiratory aerosol inhalation of the stem cell extracellular vesicle pharmaceutical composition preparation, the asthma animal model in Example 7 was used to detect it, and the specific operation was as follows:

[0202] (1) The cardiac blood of the mice at the end point was collected and coagulated at room temperature for 30 minutes. The blood was then centrifuged at room temperature (1800 g, 10 minutes) to obtain serum, which was frozen in a -80°C refrigerator for ELISA detection of relevant cytokines (IL-4, IL-5, IL-6, IL-13 and IL-17A) and IgE. Compared with dexamethasone and adipose-derived mesenchymal stem cell treatment, the adipose-derived mesenchymal stem cell extracellular vesicle drug composition preparation can reduce the levels of IL-4, IL-5, IL-6, IL-13, IL-17A and IgE in the serum of mixed granulocyte-mediated asthma mice by respiratory aerosol inhalation.

[0203] (2) Collection of end-point mouse bronchoalveolar lavage fluid (BALF): Open and fully free the mouse trachea, insert a 20G lavage needle, connect a syringe filled with 1 ml of sterile saline, and repeatedly lavage 3 times to collect as much lavage fluid as possible, then centrifuge (4°C, 500g, 20min), and take the supernatant. Resuspend the precipitate with 500μl of red blood cell lysis buffer to lyse the remaining red blood cells, centrifuge (4°C, 600g, 5min), and then resuspend the precipitate with 500μl of sterile PBS.

[0204] (3) BALF supernatant was used for ELISA to detect cytokine (IL-4, IL-5, IL-6, IL-13 and IL-17A) levels. Compared with dexamethasone and stem cell treatment, respiratory aerosol inhalation of the stem cell extracellular vesicles pharmaceutical composition preparation can reduce the levels of IL-4, IL-5, IL-6, IL-13 and IL-17A in the BALF of mixed granulocyte-mediated asthma mice.

[0205] (4) The precipitate resuspended from the bronchoalveolar lavage fluid is used in a blood cell counter to detect the total number of cells and the number of eosinophils, neutrophils, macrophages and lymphocytes.

[0206] The results showed that compared with dexamethasone and adipose-derived mesenchymal stem cell treatment, the pharmaceutical composition preparation of adipose-derived mesenchymal stem cell extracellular vesicles via respiratory nebulization inhalation could significantly reduce the level of inflammatory cell influx in the BALF of mixed granulocyte-mediated asthmatic mice.

[0207] Embodiment 11

[0208] Pharmaceutical composition preparation of stem cell extracellular vesicles can improve lung function in COPD mice

[0209] In order to explore the lung function of COPD mice after respiratory aerosol inhalation of stem cell extracellular vesicles, a nicotine-induced COPD mouse disease model was first established, and then the mice were treated and their lung function-related indicators were detected ( Fig. 9 ), the main operations are as follows:

[0210] (1) Seven-week-old female wild-type BALB / c mice were placed in an independent ventilation system in an SPF animal room for one week of adaptive feeding.

[0211] (2) BALB / c mice were placed in a nebulizer and inhaled smoke produced by burning 3 mg of nicotine through their mouths and noses. There was a 30-min interval between each two cigarettes for a total of 4 weeks.

[0212] (3) Then they were randomly divided into a control group and an experimental group, with 8 mice in each group. The control group mice were aerosolized with normal saline, while the experimental group mice received a pharmaceutical composition of human adipose-derived mesenchymal stem cell extracellular vesicles (1×10 6 The treatment was treated by aerosol inhalation of 100 μg / ml particles / mouse, once a day for 5 consecutive days.

[0213] (4) After 5 days of treatment, the mice were tested for lung function indicators, including the maximum expiratory volume in 100 ms (FEV 100 ), arterial oxygen partial pressure (PaO2), arterial carbon dioxide partial pressure (PaCO2) and oxygen saturation (SaO2).

[0214] The results showed that compared with the treatment of control group mice, the pharmaceutical composition preparation of human adipose-derived mesenchymal stem cell extracellular vesicles inhaled through the respiratory tract can significantly improve the maximum expiratory volume within 100ms of COPD model mice, increase arterial oxygen partial pressure and oxygen saturation, and reduce arterial carbon dioxide partial pressure, thereby improving their lung function.

[0215] discuss

[0216] A large number of studies have shown that mesenchymal stem cells have the effects of promoting tissue repair and anti-inflammatory effects. Most of their functions are produced by interacting with target cells through secreted soluble factors or vesicles. Mesenchymal stem cell vesicles play an important role in the treatment of many diseases and also have great application potential in the prevention and treatment of respiratory inflammatory diseases. Stem cell vesicles are important information carriers for signal exchange between stem cells and target cells. Once secreted into the extracellular space, stem cell vesicles can be engulfed by target cells through interaction with target cells, and ultimately affect the phenotype of target cells, thereby exerting their unique biological functions.

[0217] Therefore, in the present invention, human adipose-derived mesenchymal stem cells produced under GMP conditions are preferably used as parent cells for producing stem cell vesicles, and a pharmaceutical composition preparation obtained by producing the vesicles is further produced and administered via airway atomization inhalation for the treatment of acute respiratory inflammation and chronic respiratory inflammation such as bronchial asthma and chronic obstructive pulmonary disease, in order to provide a new treatment method for respiratory inflammation caused by various inducements.

[0218] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A human adipose-derived mesenchymal stem cell extracellular vesicle preparation, characterized in that: The particle size of the extracellular vesicles is 80-120nm; and the extracellular vesicles contain bioactive factors and miRNA related to anti-inflammation or tissue damage repair, the bioactive factors include matrix protein metalloenzyme 1 (MMP-1) and hepatocyte growth factor (HGF), wherein the content of MMP-1 in the preparation is 1500-2000pg / ml, the content of HGF in the preparation is 5500-6000pg / ml, and the miRNA includes hsa-let-7b-5p, hsa-let-7a-5p, hsa-miR-126-5p, hsa-miR-151a-3p, hsa-miR-1246, hsa-miR-21-5p, hsa-miR-146a-5p and hsa-let-7b-3p.

2. The preparation according to claim 1, characterized in that The stem cell extracellular vesicles are derived from the supernatant collected during the in vitro culture of human stem cells.

3. The preparation according to claim 1, characterized in that The stem cell extracellular vesicles have the following characteristics: It is prepared by polyethylene glycol (PEG) precipitation method, and the particle size of the extracellular vesicles is concentrated between 80-120nm, and the particle size is smaller and more uniform.

4. The preparation according to claim 3, characterized in that The polyethylene glycol (PEG) precipitation comprises the following steps: using PEG 3000-9000, configuring 8%-20% PEG mother solution with PBS, filtering and sterilizing, and adding the mother solution to the treated conditioned medium in a certain proportion, wherein the conditioned medium is obtained by differential centrifugation and filtering the culture supernatant of human adipose mesenchymal stem cells, and incubating at 4°C overnight; then centrifuging at 4°C, 3,000-5000g for 30-60 minutes, discarding the supernatant, adding pre-cooled PBS to resuspend the precipitate; ultra-high-speed centrifuging at 4°C, 100000-120000g for 60-120 minutes, and discarding the supernatant, thereby obtaining the stem cell extracellular vesicles.

5. The preparation according to claim 4, characterized in that The filtration sterilization is performed by filtering with a 0.22 μm filter.

6. The preparation according to claim 4, characterized in that The ratio is about 1:1 by volume.

7. A pharmaceutical composition, characterized in that The pharmaceutical composition contains: (a) the stem cell extracellular vesicle preparation according to claim 1, and (b) a pharmaceutically acceptable carrier.

8. The pharmaceutical composition according to claim 7, characterized in that The dosage form of the pharmaceutical composition is selected from the following group: aerosol inhalation, eye drops, nasal drops, and injection.

9. A method for preparing a pharmaceutical composition containing the stem cell extracellular vesicles according to claim 1, the method comprising the steps of: (S1) culturing human adipose-derived mesenchymal stem cells to a predetermined confluence of 75-90%; (S2) continuing to culture the cells for 30-60 hours under conditions suitable for producing stem cell extracellular vesicles; (S3) removing cells from the culture system by differential centrifugation and filtration, thereby isolating a culture medium containing stem cell extracellular vesicles, which is the "conditioned medium"; (S4) mixing the conditioned medium with polyethylene glycol (PEG) to form a first mixture, and placing the mixture for 12-48 hours to form stem cell extracellular vesicles modified with PEG; wherein, The polyethylene glycol is a 20% PEG9000 stock solution prepared in PBS, which is added to the treated conditioned medium at a volume ratio of 1:1; (S5) centrifuging the first mixture in the previous step at 4° C. and 4000 g for 50 minutes to precipitate the PEG-modified stem cell extracellular vesicles, and discarding the supernatant to obtain a PEG-modified stem cell extracellular vesicle precipitate; (S6) resuspending the PEG-modified stem cell extracellular vesicle precipitate obtained in the previous step to obtain a first resuspended mixture; (S7) performing ultra-high speed centrifugation on the first resuspended mixture at 4° C. and 120,000 g for 60 minutes to precipitate the PEG-modified stem cell extracellular vesicles, and discarding the supernatant to obtain a PEG-modified stem cell extracellular vesicle precipitate; (S8) resuspending the PEG-modified stem cell extracellular vesicle precipitate obtained in the previous step to obtain a medical stem cell extracellular vesicle preparation; Furthermore, the method further comprises: (S9) Mixing the medically usable stem cell extracellular vesicle preparation with a pharmaceutically acceptable carrier to prepare a pharmaceutical composition.

10. Use of the stem cell extracellular vesicle preparation according to any one of claims 1 to 6 or the pharmaceutical composition according to claim 7 for preparing a drug or preparation for treating respiratory inflammation.

11. The use according to claim 10, characterized in that The respiratory tract inflammation includes acute respiratory tract inflammation and chronic respiratory tract inflammation.

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