WO2021180237A1 - 含人体细胞衍生的细胞膜外囊泡的雾化吸入制剂、制法及其应用 - Google Patents

含人体细胞衍生的细胞膜外囊泡的雾化吸入制剂、制法及其应用 Download PDF

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WO2021180237A1
WO2021180237A1 PCT/CN2021/080869 CN2021080869W WO2021180237A1 WO 2021180237 A1 WO2021180237 A1 WO 2021180237A1 CN 2021080869 W CN2021080869 W CN 2021080869W WO 2021180237 A1 WO2021180237 A1 WO 2021180237A1
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cells
extracellular vesicles
pharmaceutical composition
peg
human
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English (en)
French (fr)
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戴成祥
李萍
王静
李苏克
雷继刚
陈应炉
宋晓乐
刘必佐
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西比曼生物科技（上海）有限公司
西比曼生物科技（无锡）有限公司
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Priority to AU2021234601A priority Critical patent/AU2021234601A1/en
Priority to EP21768139.4A priority patent/EP4134105A4/en
Priority to US17/910,444 priority patent/US20230120324A1/en
Priority to JP2022554328A priority patent/JP2023516458A/ja
Publication of WO2021180237A1 publication Critical patent/WO2021180237A1/zh

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    • A—HUMAN NECESSITIES
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    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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    • A61K9/0073—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
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    • A61P39/00—General protective or antinoxious agents
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
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    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes

Definitions

  • the present invention relates to the field of biomedicine, in particular to an atomized inhalation preparation containing extracellular vesicles derived from human cells, a preparation method and application thereof.
  • ARDS Acute respiratory distress syndrome
  • ARDS is most common in pneumonia, sepsis, aspiration of gastric contents, or severe trauma, accounting for about 10% of patients in intensive care units worldwide. Although some progress has been made in the past few decades, the mortality rate is still as high as 30-40% in most studies. In the United States, there are about 200,000 ARDS cases each year, and the hospital mortality rate is as high as 38.5%, and in the past few decades, this situation has not improved significantly.
  • Acute lung injury (ALI) and ARDS have the same pathological changes, the most common is diffuse alveolar injury.
  • the pathological basis of ALI/ARDS is the injury of alveolar epithelium and alveolar capillary endothelium caused by a variety of inflammatory cells (macrophages, neutrophils, lymphocytes, etc.) mediated local lung inflammation and uncontrolled inflammation.
  • the main pathological feature is the formation of protein-rich pulmonary edema and hyaline membrane in alveolar exudate caused by increased pulmonary microvascular permeability, which may be accompanied by pulmonary interstitial fibrosis.
  • the pathophysiological changes are dominated by decreased lung compliance, increased intrapulmonary shunt, and unbalanced ventilation blood flow ratio.
  • the clinical manifestations are respiratory frequency, respiratory distress, and refractory hypoxemia. Chest X-ray shows diffuse infiltrates of both lungs, and multiple organ failure is often complicated in the later stage.
  • ARDS ARDS based on clinical diagnostic criteria agreed by experts.
  • the focus of patient management is to implement lung protection ventilation strategies. No specific drug therapy has yet been determined.
  • the long-term prognosis of patients surviving ARDS is increasingly regarded as an important research goal, because many ARDS patients survive with sequelae such as organ function and/or cognitive psychology.
  • Future research directions include promoting the early recognition of ARDS, adding prognostic and/or predictive functions in clinical research to identify subgroups that may have therapeutic effects, and continuing efforts to understand the basic mechanisms of lung injury.
  • SARS-CoV-2 coronavirus
  • ARDS acute respiratory distress syndrome
  • SARS-CoV-2 and other coronaviruses there are currently no specific vaccines and antiviral treatments. These infectious diseases have seriously affected human life and health, and the development of effective therapeutic drugs is imminent. It is of great social significance to develop low-toxic and high-efficiency drugs for diseases such as SARS-CoV-2 and other coronaviruses such as pneumonia and ARDS to meet the clinical needs of patients at home and abroad.
  • the purpose of the present invention is to provide a high-efficiency, low-toxicity, safe and mass-produced medicine for treating diseases such as ARDS.
  • a pharmaceutical composition in the first aspect of the present invention, contains (a) an active substance derived from human somatic cells, and the active substance is an extracellular capsule produced by human somatic cells Vesicles; and (b) a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is a cell-free pharmaceutical composition.
  • the "cell-free" means that the pharmaceutical composition does not contain live cells and dead cells.
  • the somatic cells are selected from the group consisting of stem cells, progenitor cells, immune cells, or combinations thereof derived from human tissue, bone marrow and/or blood.
  • the dosage form of the pharmaceutical composition is selected from the group consisting of a liquid dosage form and a solid dosage form (such as a lyophilized dosage form).
  • the dosage form of the pharmaceutical composition is selected from the group consisting of aerosol inhalation, eye drops, and nasal drops.
  • the pharmaceutical composition has the following characteristics:
  • (P1) Better storage stability than living cell preparations; preferably, the storage includes storage at room temperature and/or low temperature (such as -196°C to 25°C, preferably -100°C to 0°C);
  • the "storage stability better than living cell preparations” means that the stability of the pharmaceutical composition is better than the stability of living cell preparations under the same conditions;
  • (P2) High dispersibility; preferably, when the pharmaceutical composition is an aqueous liquid preparation (such as a solution prepared with physiological saline), it is placed at 0-25°C for 6-24 hours, and it appears colorless and transparent. And there is no visible flocs and sediments.
  • an aqueous liquid preparation such as a solution prepared with physiological saline
  • the pharmaceutical composition is applied to a site other than the skin.
  • the pharmaceutical composition is applied to a site selected from the group consisting of eyeball conjunctiva, eyelid conjunctiva, retina, oral cavity, nasal cavity, upper respiratory tract, lower respiratory tract, gastrointestinal tract, lung, or combination.
  • the pharmaceutical composition is administered to cells selected from the group consisting of vascular endothelial cells, alveolar type 1 epithelial cells, alveolar type 2 epithelial cells, monocytes macrophages, neutrophils, Dendritic cells, antigen presenting cells, T lymphocytes, fibroblasts, central nerve cells, peripheral nerve cells and peripheral nerve fibers, or combinations thereof.
  • the stem (progenitor) cells are selected from the group consisting of mesenchymal stem cells derived from human adipose tissue, alveolar epithelial progenitor cells derived from human lung tissue, and mesenchymal stem cells derived from human umbilical cord Wharton’s glue.
  • Stem cells mesenchymal stem cells derived from human endometrial tissue, stem cells derived from human placental tissue, epithelial cells derived from human placental amniotic membrane, mononuclear cells in human blood, hematopoietic stem cells in human bone marrow tissue, and human bone marrow tissue
  • Mesenchymal stem cells derived from human periosteum keratinocytes derived from human skin tissue, dendritic cells derived from human blood tissue, CD4-positive T lymphocytes derived from human blood tissue, and platelets derived from human blood tissue , Or a combination thereof.
  • the extracellular vesicles include nanovesicles surrounded by a lipid bilayer membrane structure.
  • the diameter of the nanovesicles is between 30-1000 nanometers.
  • the nanovesicles contain different types of microribonucleic acid (miRNA), small ribonucleic acid (sRNA), non-coding DNA fragments, and transfer ribonucleic acid (t-RNA) with specific functions. ), soluble cytokines, growth factors, and other proteins.
  • miRNA microribonucleic acid
  • sRNA small ribonucleic acid
  • t-RNA transfer ribonucleic acid
  • soluble cytokines soluble cytokines
  • growth factors and other proteins.
  • the soluble active cytokine is selected from the following group: soluble free lipoprotein molecules, glycoprotein molecules, biosignal molecules, tissue growth factor ⁇ (TGF ⁇ ), hepatocyte growth factor (HGF) , Vascular Endothelial Cell Growth Factor (VEGF), Epidermal Growth Factor (KGF), Interleukin-10 (IL-10), Interleukin-1 ⁇ Receptor Antagonist (IL-1 ⁇ A), or a combination thereof.
  • TGF ⁇ tissue growth factor ⁇
  • HGF hepatocyte growth factor
  • VEGF Vascular Endothelial Cell Growth Factor
  • KGF Epidermal Growth Factor
  • IL-10 Interleukin-10
  • IL-1 ⁇ A Interleukin-1 ⁇ Receptor Antagonist
  • the soluble active cytokine is a soluble active cytokine produced by the human somatic cells.
  • the soluble active cytokine includes the active cytokine produced by paracrine of human somatic cells cultured in the natural state, and the active cytokine produced by the expression of human somatic cells modified with exogenous genes. , Or a combination thereof.
  • the extracellular vesicles specifically express the following proteins: CD9, CD63, CD81 and TSG101; they do not express or substantially do not express the protein CANX.
  • the diameter of the extracellular vesicles is preferably 50-500 nm.
  • the diameter is an average diameter.
  • the cells used to produce the extracellular vesicles include cells from the following sources:
  • the cells used to produce the extracellular vesicles include primary cells and passage cells with 1-10 passages.
  • the cells used to produce the extracellular vesicles include cells that have not been genetically manipulated, and cells that have been genetically manipulated.
  • the gene manipulation includes gene editing, gene introduction, gene knock-down, gene knock-out, or a combination thereof.
  • the cells used to produce the extracellular vesicles are pretreated cells.
  • the somatic cells are adipose-derived mesenchymal stem (progenitor) cells, placental amniotic membrane-derived mesenchymal stem cells, or a combination thereof.
  • the fat-derived mesenchymal stem (progenitor) cells are obtained through the following methods: for those that have undergone ethical review and record, signed informed consent, and undergo strict laboratory inspections to meet the requirements of fatty stem ( Progenitor) cell products are built into the standard male or female healthy volunteers, after abdominal wall skin puncture liposuction, or abdominal wall surgery to cut fat, stored in the cell preservation solution, transported to a qualified GMP laboratory at low temperature, and separated , Purification, proliferation, and the obtained adipose-derived mesenchymal progenitor cells, that is, working bank cells (intermediate products).
  • the mesenchymal stem (progenitor) cells derived from human adipose tissue are produced in a GMP production laboratory in accordance with the intermediate product production process, and meet the negative standards of various pathogenic tests.
  • Specific surface markers such as CD73-positive, CD90-positive, CD105-positive cell percentage is about 98%, and the percentage of CD34-positive/CD45-positive and HLA-DR-positive cells is less than 2%, which meets the standards for antibiotic residue and serum and serum substitute residue detection standards , P1-P6 generation adipose mesenchymal stem (progenitor) cells.
  • the adipose-derived mesenchymal stem (progenitor) cells are P3-P4 generation adipose-derived mesenchymal stem (progenitor) cells.
  • the somatic cells are stored at -196°C to -80°C (preferably, -196°C to -135°C) for 0-36 months (preferably 0-135°C).
  • Mesenchymal stem (progenitor) cells derived from adipose tissue at 24 months or 0.5-24 months).
  • the production process of the extracellular vesicles and soluble active cytokines derived from human somatic cells includes the steps:
  • the culture conditions are 37 ⁇ 1°C, 5 ⁇ 0.5% CO 2 , to the degree of cell fusion Reach 80 ⁇ 10%, and then use a specific pretreatment medium for culture, the culture condition is 37 ⁇ 1°C, 5 ⁇ 0.5% CO 2 , and culture for 36-72 hours;
  • the differential centrifugation combined with polyethylene glycol PEG precipitation includes the steps of: using PEG3000-PEG9000 PEG, configuring 8%-30% PEG with PBS, and sterilizing through filtration (such as After filtering with a 0.22 ⁇ m filter), add it to the treated conditioned medium according to a certain ratio (for example, about 1:1 volume ratio), and incubate overnight at 4°C. Centrifuge at 3,000-5000g for 30-60 minutes at 4°C, and discard the supernatant.
  • the extracellular vesicles obtained by the production process have the following biomarker characteristics: CD9 positive (ie CD9 + ), CD63 positive, CD81 positive, TSG101 positive, and CANX negative.
  • the production process has the following characteristics: it has a clinical grade cell product production scale under GMP laboratory conditions.
  • the clinical-grade production scale means that one production operation unit can separate 800-1200ml conditioned medium obtained from two cell factories, and 2-5 ⁇ 10 11 extracellular vesicles can be separated. Calculated based on the total dose of 2-5 ⁇ 10 9 extracellular vesicles used locally for a patient, one production operation unit and one batch production can meet the local consumption of 100-250 patients. Or, with 100 production operation units, a batch of production can reach a total of 10,000-25,000 people, and it will take 5-6 days.
  • the feature of the production process is: the separated extracellular vesicles, soluble active cytokines and PEG of a specific molecular weight are cross-linked to form hydrophilic particles with a specific particle size (for example, less than 3 microns). ,
  • a specific particle size for example, less than 3 microns.
  • the extracellular vesicles, soluble active cytokines, and polyethylene glycol PEG cross-linked hydrophilic particles are dispersed in isotonic sodium chloride solution, low molecular hyaluronic acid solution, artificial tears , Gel solution, so as to make atomized inhalation liquid, eye drops, nasal drops, and external gel preparations.
  • the pharmaceutical composition is applied to mucosal cells, and the mucosal cells can take up extracellular vesicles.
  • the pharmaceutical composition of the present invention is an aerosol inhaler.
  • the pharmaceutical composition of the present invention (especially aerosol inhaler) is used to treat infectious lung injury, preferably for viral lung injury, and more preferably for coronavirus (such as SARS).
  • coronavirus such as SARS
  • the pharmaceutical composition is an aerosol prepared from adipose tissue-derived progenitor cells and placental amniotic membrane-derived mesenchymal stem cells derived extramembranous vesicles and soluble cytokines and isotonic sodium chloride solution Inhale liquid.
  • the pharmaceutical composition is used to treat diseases caused by viruses.
  • the virus is selected from the group consisting of influenza virus, SARS coronavirus, SARS coronavirus 2, MERS coronavirus infection.
  • the pharmaceutical composition (such as aerosolized inhalation liquid) is used for the treatment of viral acute lung injury, through the aerosolized inhalation treatment, so as to prevent the release of acute lung injury inflammatory factors and reduce the height of the alveolar.
  • the infiltration of protein liquid and the damage of alveolar epithelial cells significantly increase the rescue success rate of patients with acute lung injury, and improve the lung function and quality of life of surviving patients.
  • a method for preparing extracellular vesicles which includes the steps:
  • T1 is usually 24-72 hours, preferably 30-60 hours;
  • S4 Mix the conditioned medium and polyethylene glycol (PEG) to form a first mixture, and leave it for a period of T2 to form PEG-modified extracellular vesicles; wherein, The T2 mentioned is usually 6-60 hours, preferably 12-48 hours;
  • the method further includes:
  • the medicinal extracellular vesicle preparation (or active substance) is mixed with a pharmaceutically acceptable carrier to prepare a pharmaceutical composition.
  • the method further includes preparing the pharmaceutical composition into an aerosol inhalation preparation, an injection, or a lyophilized preparation.
  • an extracellular vesicle preparation is provided, and the extracellular vesicle preparation is prepared by the method described in the second aspect of the present invention.
  • the extracellular vesicle preparation includes nanovesicles surrounded by a lipid bilayer membrane structure.
  • the diameter of the nanovesicles is between 30-1000 nanometers.
  • the nanovesicles contain different types of microribonucleic acid (miRNA), small ribonucleic acid (sRNA), non-coding DNA fragments, and transfer ribonucleic acid (t-RNA) with specific functions. ), soluble cytokines, growth factors, and other proteins.
  • miRNA microribonucleic acid
  • sRNA small ribonucleic acid
  • t-RNA transfer ribonucleic acid
  • soluble cytokines soluble cytokines
  • growth factors and other proteins.
  • the soluble active cytokine is selected from the following group: soluble free lipoprotein molecules, glycoprotein molecules, biosignal molecules, tissue growth factor ⁇ (TGF ⁇ ), hepatocyte growth factor (HGF) , Vascular endothelial cell growth factor (VEGF), epidermal cell growth factor (KGF), interleukin-10 (IL-10), interleukin-1 ⁇ receptor antagonist (IL-1 ⁇ A), or a combination thereof.
  • TGF ⁇ tissue growth factor ⁇
  • HGF hepatocyte growth factor
  • VEGF Vascular endothelial cell growth factor
  • KGF epidermal cell growth factor
  • IL-10 interleukin-10
  • IL-1 ⁇ A interleukin-1 ⁇ receptor antagonist
  • the soluble active cytokine is a soluble active cytokine produced by the human somatic cells.
  • the soluble active cytokine includes the active cytokine produced by paracrine of human somatic cells cultured in the natural state, and the active cytokine produced by the expression of human somatic cells modified with exogenous genes. , Or a combination thereof.
  • the extracellular vesicles specifically express the following proteins: CD9, CD63, CD81 and TSG101; they do not express or substantially do not express the protein CANX.
  • the fourth aspect of the present invention there is provided a use of the pharmaceutical composition according to the first aspect of the present invention or the extracellular vesicle preparation according to the third aspect of the present invention, which are used for the preparation of prevention and/or treatment of inflammation, Or injury drugs.
  • the inflammation is selected from the group consisting of viral infectious inflammation, bacterial infectious inflammation, fungal infectious inflammation, autoimmune inflammation, or a combination thereof;
  • the injury is selected from the following group: ischemic injury, hypoxic injury, chemical injury, physical injury, or a combination thereof.
  • the drug is used to treat diseases caused by viruses.
  • the virus is selected from the group consisting of influenza virus, SARS coronavirus, SARS coronavirus 2, MERS coronavirus infection.
  • a method for preventing and/or treating inflammation or injury including the steps of: administering the pharmaceutical composition according to the first aspect of the present invention or the third aspect to a subject in need The above-mentioned extracellular vesicle preparation.
  • the inflammation is selected from the group consisting of viral infectious inflammation, bacterial infectious inflammation, fungal infectious inflammation, autoimmune reactive inflammation, or a combination thereof.
  • the injury is selected from the group consisting of ischemic injury, hypoxic injury, chemical injury, physical injury, or a combination thereof.
  • the drug is used to treat acute respiratory distress syndrome.
  • the medicine is an atomized inhalation liquid.
  • the drug is used to treat viral acute lung injury.
  • the drug is treated by aerosol inhalation, so as to prevent the release of inflammatory factors in acute lung injury, reduce the infiltration of high-protein liquid in the alveoli and the damage of alveolar epithelial cells.
  • the subject is a human.
  • Figure 1 shows the production of extracellular vesicles collected by Western blotting and NTA detection by PEG precipitation and ultracentrifugation.
  • Figure (1A) M is Marker; 1 to 4 are respectively 8% PEG 6000, 12% PEG 6000, 16% PEG 6000, 20% PEG 6000 isolated CD63 expression level of extracellular vesicles (the same amount of protein Loading);
  • Figure (1D) NTA detects the size of the extracellular vesicles separated by PEG6000 and ultracentrifugation; *p ⁇ 0.05.
  • Figure 2 shows the uptake of extracellular vesicles by fibroblasts. Among them, blue: nucleus stained by Hochest; green: extracellular vesicles labeled with PKH-67; ruler: 100 ⁇ m.
  • Figure 3 shows the number of extracellular vesicles stored at 4°C, -20°C and -80°C for 9 weeks.
  • Figure 4 shows the expression of CD81, a specific marker of extracellular vesicles stored at 4°C, -20°C and -80°C for 9 weeks.
  • Figure 5 shows the state of extracellular vesicles under the transmission electron microscope after half an hour of re-fusion at room temperature.
  • Figure 5A shows the state of extracellular vesicles stored at -80°C for 1 week under transmission electron microscopy
  • Figure 5B shows the state of extracellular vesicles under transmission electron microscopy after refusion at room temperature for half an hour.
  • Figure 6 shows the particle size concentration and size of extracellular vesicles after half an hour of re-fusion at room temperature.
  • Figure 7 shows that extracellular vesicles of human adipose-derived mesenchymal progenitor cells inhibit LPS-induced macrophage activation.
  • A Pro-inflammatory macrophage morphological observation
  • B Pro-inflammatory macrophage morphological statistical analysis.
  • Control is the control group
  • LPS is the macrophage treatment group with lipopolysaccharide alone
  • LPS+Dex is the lipopolysaccharide and dexamethasone treatment group
  • LPS+haMPCs-Exo is the lipopolysaccharide and adipose mesenchymal progenitor cell extramembrane vesicle treatment group
  • the red arrow indicates activated macrophages (pro-inflammatory macrophages); *P ⁇ 0.05, the scale is 50 ⁇ m.
  • Figure 8 shows that extramembrane vesicles of human adipose-derived mesenchymal progenitor cells inhibit LPS-induced macrophage pro-inflammatory factor gene expression.
  • (A), (B) and (C) respectively show real-time fluorescent quantitative PCR to detect the gene expression levels of macrophage inflammatory factors TNF- ⁇ , IL-1 ⁇ and IL-6.
  • Control is the control group
  • LPS is the macrophage treatment group with lipopolysaccharide alone
  • LPS+Dex is the lipopolysaccharide and dexamethasone treatment group
  • LPS+haMPCs-Exo is the lipopolysaccharide and adipose mesenchymal progenitor cell extramembrane vesicle treatment group ;***P ⁇ 0.001.
  • Figure 9 shows that extramembrane vesicles of human adipose-derived mesenchymal progenitor cells inhibit LPS-induced macrophage pro-inflammatory factor release.
  • LPS induces macrophages and is processed by human adipose-derived mesenchymal progenitor cells with extra-membrane vesicles.
  • B ELISA detects the levels of inflammatory factors IL-6 and TNF- ⁇ released by macrophages.
  • Control is the control group
  • LPS is the macrophage treatment group with lipopolysaccharide alone
  • LPS+Dex is the lipopolysaccharide and dexamethasone treatment group
  • LPS+haMPCs-Exo is the lipopolysaccharide and adipose mesenchymal progenitor cell extramembrane vesicle treatment group ; **P ⁇ 0.01, ***P ⁇ 0.001.
  • Figure 10 is a schematic diagram of extracellular vesicles, soluble cytokines and PEG cross-linked to form hydrophilic particles.
  • FIG 11 shows a schematic process route (from step S4 to S8) of the present invention.
  • the biomedical preparation contains extracellular vesicles derived from human cells. Specific particle size and modified with specific PEG, so it can not only be stored stably for a long time, but also has excellent dispersibility in medical solvents (such as physiological saline, etc.). It is especially suitable for direct application to patients through aerosol inhalation.
  • the present invention has been completed on this basis.
  • Coronavirus belongs to the Nidovirales (Nidovirales) Coronaviridae (Coronaviridae), which is an enveloped positive-stranded RNA virus, and its subfamily includes four genera of ⁇ , ⁇ , ⁇ and ⁇ .
  • HCoV-229E and HCoV-NL63 belong to the ⁇ genus coronavirus
  • HCoV-OC43, SARS-CoV, HCoV-HKU1, MERS-CoV and SARS-CoV-2 are all ⁇ genus coronavirus Virus.
  • the new coronavirus (SARS-CoV-2) that broke out at the end of 2019 has about 80% similarity with SARS-CoV and 40% similarity with MERS-CoV, and it also belongs to the beta coronavirus.
  • EVs extracellular vesicles
  • exosomes lipid bilayers with diameters ranging from 30-2000nm.
  • microvesicles lipid bilayers with diameters ranging from 30-2000nm.
  • exosomes refers to a subclass of EVs with a diameter of 50-100 nm derived from endosomes. They are the main paracrine secretions of various cell types including mesenchymal stem cells (MSCs). component.
  • MSCs mesenchymal stem cells
  • MSCs exosomes are a type of MSCs-derived EVs with a diameter in the range of 50-100nm and a complete lipid bilayer membrane structure.
  • Exosomes are a kind of carriers that carry abundant goods, and their function is mainly through the continuous transfer of microribonucleic acid (miRNAs) and proteins.
  • miRNAs microribonucleic acid
  • proteins proteins that can change the various activities of target cells through different ways.
  • MSC exosomes are involved in 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) and other physiological and pathological processes.
  • MSC exosomes contain some cytokines and growth factors, such as TGF ⁇ 1, interleukin-6 (IL-6), IL-10 and hepatocyte growth factor (HGF), etc. These factors have been confirmed to have Helps immune regulation.
  • cytokines and growth factors such as TGF ⁇ 1, interleukin-6 (IL-6), IL-10 and hepatocyte growth factor (HGF), etc. These factors have been confirmed to have Helps immune regulation.
  • VEGF vascular endothelial growth factor
  • EMMPRIN extracellular matrix metalloproteinase inducer
  • MMP-9 have been reported in MSC exosomes. These three proteins play an important role in stimulating angiogenesis. It may be the basis of the repair function of exosomes.
  • NIR near-infrared
  • EVs labeled with superparamagnetic iron oxide nanoparticles have high-resolution and sensitive magnetic resonance analysis capabilities, which provide a basis for accurate detection of deep organs.
  • DiI-labeled MSC exosomes can be shown to reach the brain, liver, lung and spleen.
  • DID-labeled EVs specifically accumulate in the kidneys of AKI mice, indicating that exosomes seem to be able to home to the injury site.
  • EVs Compared with intravenous injection, nasal administration can better accumulate brain exosomes at the injury site.
  • the biodistribution of EVs through systemic administration is a dynamic process: within about 30 minutes after administration, EVs rapidly distributes in the liver, spleen and lungs, and then is processed by the liver and kidneys to enter the elimination stage, from 1 to 1 after administration. Clear EVs within 6 hours.
  • exosomes play a key role in regulating tumor-specific T cell activation by carrying and presenting functional MHC peptide complexes.
  • Exosomes released from bone marrow-derived MSCs can effectively improve the chronic transplantation of mice by inhibiting the activation and infiltration of CD4-positive T cells, reducing the production of pro-inflammatory cytokines, promoting the production of IL-10-expressing Tregs, and inhibiting Th17 cells.
  • Host-resistant disease (cGVHD) Host-resistant disease
  • EVs derived from human pluripotent stromal cells can inhibit autoimmunity in type 1 diabetes (T1D) and experimental autoimmune uveoretinitis (EAU) models. EVs can inhibit the activation of antigen-presenting cells (APC), inhibit the development of Th1 and Th17 cells, and increase the expression of the immunosuppressive factor IL-10.
  • T1D type 1 diabetes
  • EAU experimental autoimmune uveoretinitis
  • Human bone marrow MSCs exosomes can promote the proliferation of regulatory T cell subsets and up-regulate the expression of cytokines IL-10 and TGF- ⁇ 1 in peripheral blood mononuclear cells (PBMCs) to enhance the immunosuppressive ability of asthma patients.
  • PBMCs peripheral blood mononuclear cells
  • miR-181c exerts an anti-inflammatory effect in the inflammation model of burn rats by down-regulating the TLR4 signaling pathway.
  • a single intrarenal injection of porcine autologous fat MSCs-EVs can reduce the levels of multiple pro-inflammatory cytokines (TNF- ⁇ , IL-6, IL-1- ⁇ ) in the renal vein of a pig model of renal artery stenosis, and inhibit inflammation Increased levels of factor IL-10, accompanied by the migration of macrophages from pro-inflammatory cells to repair macrophages, once again proved the immunomodulatory potential of EVs.
  • TNF- ⁇ , IL-6, IL-1- ⁇ pro-inflammatory cytokines
  • Adipose MSCs exosomes can significantly reduce the increase in serum alanine aminotransferase and aspartate aminotransferase levels in the C57BL/6 mouse hepatitis model induced by Concanavalin A (Con A), reduce liver inflammation, and reduce the hepatitis model
  • the levels of pro-inflammatory cytokines (TNF- ⁇ , IFN- ⁇ , IL-6, IL-18, IL-1 ⁇ ) in mouse serum inhibited the activation of inflammasome in the liver of model mice.
  • Sepsis is a systemic inflammatory response caused by the body's fight against microbial infections. Despite the application of advanced antibiotics, the mortality rate of sepsis in the intensive care unit is still high, so researchers have targeted MSCs for treatment of such systemic inflammatory diseases.
  • MSC-EVs in animal models of sepsis caused by cecal ligation has been extensively studied. In a rat model of sepsis, intravenous administration of adipose MSCs-EVs alleviated systemic inflammation, organ damage and subsequent lethality.
  • the efficacy of EVs produced from MSCs pretreated with IL-1 ⁇ to treat sepsis was significantly higher than that of EVs derived from MSCs without pretreatment.
  • the study also shows that EVs obtained from induced MSCs can effectively polarize macrophages and differentiate them into M2 type, which is the anti-inflammatory phenotype of macrophages.
  • M2 type which is the anti-inflammatory phenotype of macrophages.
  • miR-146a the content of miR-146a in MSCs and EVs pretreated with IL-1 ⁇ is significantly increased.
  • Transfer of miR-146a packaged in EVs into macrophages can polarize it to M2 type [Casado JG, et al. Frontiers in Veterinary Science. 2017; 4:39]
  • AT2R-MSCs AT2R-MSCs
  • LPS lipopolysaccharide
  • MSCs exosomes alleviate lung ischemia/reperfusion injury in mice by transporting anti-apoptotic miR-21-5p.
  • the miR-21-5p in exosomes reduces apoptosis induced by oxidative stress by targeting PTEN and PDCD4 in lung tissue.
  • exosomal miR-146a enhanced the therapeutic effect of IL-1 ⁇ pretreated MSC. It was further found that IL-1 ⁇ stimulated MSCs, which could up-regulate the expression of MSCs miR-146a, the latter Can be packaged in exosomes. Then, this exosome miR-146a was transferred to macrophages, resulting in M2 polarization, which eventually led to an increase in the survival rate of septic mice. Therefore, the use of specific miRNA overexpression to modify the exosomes of MSCs is a promising new direction for the development of ARDS treatment.
  • ARDS is an acute systemic inflammatory response caused by direct or indirect lung injury caused by factors such as smoking, drowning, aspiration, sepsis, trauma, ischemia, and exposure to toxins. Severe inflammation causes changes in vascular permeability, leading to acute pulmonary edema.
  • the pathological process of ARDS has three main stages: exudative phase, proliferative phase and fibrotic phase.
  • the inflammatory cascade caused by lung injury and the dysfunction of the alveolar-capillary barrier leads to an increase in the permeability of alveolar epithelium and pulmonary capillary endothelial cells, which are the characteristics of the exudative phase.
  • the pathological manifestations of lung tissue are diffuse alveolar injury with exudation, microvascular injury with secondary pulmonary edema, necrosis of alveolar type 1 (AT1) epithelial cells, aggregation of inflammatory cells, and release of active mediators.
  • Alveolar inflammation is mainly caused by polymorphonuclear neutrophils, monocytes and macrophages.
  • Other pro-inflammatory mechanisms are also involved, such as lung cells, inflammatory cells and fibroblasts that release large amounts of pro-inflammatory cytokines.
  • a transparent membrane is formed in the alveoli, accompanied by the infiltration of inflammatory cells, including T lymphocytes, neutrophils and macrophages.
  • inflammatory cells including T lymphocytes, neutrophils and macrophages.
  • extracellular matrix is deposited in the alveoli, accompanied by continuous chronic inflammation.
  • the inflammatory cascade plays a key role in the process of apoptosis, proliferation, migration and other processes closely related to ARDS. Continuous damage and damage that cannot be repaired in time are the main pathological manifestations of ARDS fiber proliferation.
  • fibroblast proliferation there are fibroblast proliferation, AT2 cell proliferation and lung tissue repair.
  • the repair mechanism of damaged alveolar epithelium is not fully understood. It includes the proliferation of AT2 cells. AT2 cells migrate along the basement membrane to form a new epithelial barrier, and interact with extracellular matrix and other cells including alveolar macrophages. Effect, leading to significant changes in lung structure and function in some cases. Computed tomography (CT) of the lungs can detect dense fibrosis and honeycomb structures during the fibrous proliferation phase of ARDS.
  • CT Computed tomography
  • Severe diseases associated with ARDS can cause a high risk of ventilator-acquired pneumonia, acute myocardial infarction, and acute pulmonary embolism.
  • the invention provides an extracellular vesicle derived from mesenchymal stem cells (including adipose-derived mesenchymal stem progenitor cells, umbilical cord mesenchymal stem cells and placental amnion mesenchymal stem cells).
  • mesenchymal stem cells including adipose-derived mesenchymal stem progenitor cells, umbilical cord mesenchymal stem cells and placental amnion mesenchymal stem cells.
  • the extracellular vesicles prepared by the specific optimized process of the present invention have a specific particle size distribution and are cross-linked with PEG to form hydrophilic particles, which are not only extremely suitable for application and action in the form of aerosols In the lungs (especially the lower respiratory tract), it can exist stably in aqueous solution for a long time, and has excellent dispersibility (better than the cells themselves).
  • FIG. 10 For ease of understanding, the applicant provides a schematic diagram 10. As shown in Figure 10, representative extracellular vesicles, soluble cytokines and PEG are cross-linked to form hydrophilic particles.
  • the extracellular vesicles obtained by the separation method and process described in the present invention have a particle size in the range of 50-150 nm.
  • the extracellular vesicles, soluble cytokines and PEG are cross-linked to form hydrophilic particles with a particle size of less than 3 microns (such as about 1-3 microns or 0.5-3 microns), and the stability and dispersibility in aqueous solutions are better than the cells themselves.
  • soluble cytokines and PEG cross-linked to form hydrophilic particles respectively formulated with physiological saline, artificial tears, hyaluronic acid and other solutions for atomized inhalation, eye drops, nasal drops and external use , Is conducive to local mucosal cells to absorb and utilize these extracellular vesicles and soluble active cytokines. Its biological distribution in the body is consistent with that of simple extramembranous vesicles.
  • mesenchymal stem cells are preferably used to prepare cells of extracellular vesicles.
  • Representative examples of mesenchymal stem cells include (but are not limited to): adipose-derived mesenchymal stem cells, umbilical cord mesenchymal stem cells, placental amniotic mesenchymal stem cells, or combinations thereof.
  • the present invention also provides a pharmaceutical composition containing (a) an active substance derived from human somatic cells, and the active substance is an extracellular vesicle produced by human somatic cells; and (b) ) A pharmaceutically acceptable carrier.
  • the pharmaceutical composition is a cell-free pharmaceutical composition.
  • the active ingredients also include soluble active cytokines, and these soluble active cytokines are mainly present in vesicles outside the cell membrane.
  • these soluble active cytokines may exist outside the extracellular vesicles.
  • Some soluble active cytokines can also be modified by PEG.
  • a particularly preferred pharmaceutical preparation is an aerosol inhalation preparation.
  • the invention also provides a method for preparing extracellular vesicles.
  • a typical method is to use mesenchymal stem cells (including adipose mesenchymal stem (progenitor) cells, umbilical cord mesenchymal stem cells and placental amniotic mesenchymal stem cells) as raw materials to prepare the extracellular vesicles of the present invention.
  • mesenchymal stem cells including adipose mesenchymal stem (progenitor) cells, umbilical cord mesenchymal stem cells and placental amniotic mesenchymal stem cells
  • a typical preparation method includes the following steps:
  • S1 Culture cells (such as adipose mesenchymal stem (progenitor) cells) to reach a predetermined confluence (such as 75-90%);
  • T1 is usually 24-72 hours, preferably 30-60 hours;
  • S4 Mix the conditioned medium and polyethylene glycol (PEG) to form a first mixture, and leave it for a period of T2 to form PEG-modified extracellular vesicles; wherein, The T2 is usually 6-60 hours, preferably 12-48 hours;
  • FIG. 11 A schematic process route from step S4 to S8 is shown in FIG. 11.
  • a HyperFlask cell factory can produce 500ml conditioned medium of extracellular vesicles, and it is expected to separate 5 ⁇ 10 10-10 ⁇ 10 10 total extracellular vesicles.
  • one production operation unit in the laboratory can separate two cell factories with a total of 800-1200ml extracellular vesicles of conditioned medium. It is expected to separate 2-5 ⁇ 10 11 extracellular vesicles, according to a patient’s 2-5 ⁇ 109 Usage, one production operation unit can meet the usage of 100-250 patients in one batch.
  • a HyperFlask cell factory can produce 500-600ml conditioned medium extracellular vesicles, and it is expected to separate 2-5 ⁇ 10 11 total extracellular vesicles.
  • one production operation unit can separate 800-1200ml extracellular vesicles of conditioned medium produced by two cell factories, and it is expected that 2-5 ⁇ 10 11 extracellular vesicles can be separated and purified. According to a local dosage of 2-5 ⁇ 10 9 for a patient, one production operation unit can meet the usage of 100-250 patients in one batch.
  • hydrophilic particles of the pharmaceutical composition of the present invention that are uniformly dispersed in an aqueous solution and smaller than 3 microns can be administered by aerosol inhalation and can enter the respiratory tract, especially the lower respiratory tract, with unusually high efficiency.
  • Adipose-derived mesenchymal stem (progenitor) cells For male or female healthy volunteers who have undergone ethical review and record filing, signed informed consent, and passed strict laboratory inspections to meet the criteria for building adipose tissue stem cell products into the group, they will undergo abdominal wall puncture and aspiration. Fat, or abdominal wall surgery to cut fat, place it in the cell preservation solution and transport it to a qualified GMP laboratory at low temperature. After separation, purification, and proliferation, the adipose mesenchymal progenitor cells obtained are the working bank cells (middle product).
  • the ⁇ MEM containing 5% EliteGro was centrifuged at 120,000g for 6h at 4°C.
  • the medium is changed and the pretreated medium is used for culturing at 37°C, 5% CO 2 , and culturing for 48 hours.
  • the culture supernatant was collected, centrifuged at 3,000g for 15 minutes, then the supernatant was taken out, and centrifuged at 10,000g for 30 minutes at 4°C. Take the supernatant, which is the culture medium containing extracellular vesicles, which is referred to as "conditioned medium” hereinafter.
  • extracellular vesicles were prepared by PEG precipitation method and ultracentrifugation method, respectively.
  • Example 1 Take the conditioned medium (Example 1), ultracentrifuge at 120,000g for 70 minutes at 4°C, resuspend it in pre-cooled PBS, and resuspend the pellet with an appropriate amount of isotonic sodium chloride solution after ultracentrifugation at 120,000g for 70 minutes to obtain Extracellular vesicles.
  • Example 1 Prepared with PBS at a concentration of 8%, 12%, 16%, or 20% PEG6000, filtered with a 0.22 ⁇ m filter, and added to the treated conditioned medium (Example 1) at a volume ratio of 1:1, and placed After overnight incubation at 4°C.
  • extracellular vesicles obtained by using 12% PEG 6000 to separate 200ml medium and the extracellular vesicles obtained from 350ml medium by ultracentrifugation were loaded according to the same volume and the same amount of protein for CANX, TSG101 and CD81 identification And NTA detection.
  • NTA results showed that the concentration of extracellular vesicles isolated by PEG 6000 was 1.35 ⁇ 10 8 extracellular vesicles/ml medium, and the concentration of extracellular vesicles isolated by ultracentrifugation was 6.8 ⁇ 10 7 extracellular vesicles/ ml medium.
  • the size of the extracellular vesicles separated by PEG 6000 was 126.6 ⁇ 2.09nm, and the size of the extracellular vesicles separated by ultracentrifugation was 137.8 ⁇ 3.8nm.
  • Figure 2 shows that the extracellular vesicles isolated by PEG 6000 do not affect the uptake of extracellular vesicles by fibroblasts.
  • the separated and prepared extracellular vesicles were prepared into aqueous solutions with physiological saline and placed at 4°C, -20°C and -80°C, respectively, and stored for 9 weeks. After 9 weeks, the extracellular vesicles placed at 3 temperatures were tested for NTA and related performance tests.
  • test samples include:
  • Sample 1 Freeze-dried powder of basic medium (negative control), stored at 4°C for 4 weeks;
  • Sample 2 Freeze-dried powder of stem cell culture solution (positive control), stored at 4°C for 4 weeks;
  • Sample 3 Freeze-dried powder of extracellular vesicles, stored at room temperature for 4 weeks;
  • Sample 4 Freeze-dried powder of extracellular vesicles, stored at 4°C for 4 weeks;
  • Sample 5 Freeze-dried powder of extracellular vesicles, stored at -20°C for 4 weeks;
  • Extramembrane vesicles of human adipose-derived mesenchymal progenitor cells inhibit LPS-induced macrophage activation
  • LPS lipopolysaccharide
  • control without any drug treatment
  • 0.1 ⁇ g/ml LPS alone treatment group the 0.1 ⁇ g/ml LPS and 5 ⁇ g/ml dexamethasone (Dex) co-treatment group
  • Extramembrane vesicles of human adipose-derived mesenchymal progenitor cells inhibit LPS-induced macrophage pro-inflammatory factor gene expression levels
  • real-time fluorescent quantitative PCR was used to detect the expression of its main inflammatory factor genes level.
  • the specific operation method is basically the same as that described in Example 1, but the drug treatment time is 24 hours.
  • the total RNA extraction method is carried out using the Biyuntian RNA extraction kit instructions, while the real-time fluorescent quantitative PCR detection uses the TaqMan probe method for simultaneous detection of target genes and internal reference genes, and relative quantitative analysis according to the 2- ⁇ CT method.
  • Extramembrane vesicles of human adipose-derived mesenchymal progenitor cells inhibit LPS-induced macrophage release of pro-inflammatory factors
  • the level of inflammatory factors secreted by them was detected by ELISA.
  • the specific operation method is the same as that described in Example 2, but the drug treatment time is 24 hours. See Figure 9A for the experimental scheme.
  • the ELISA method to detect macrophage inflammatory factors was performed in accordance with the instructions of the Biyuntian TNF- ⁇ and IL-6 kit, and then statistical analysis was performed.
  • TNF- ⁇ and IL-6 levels were also extremely significantly reduced, reaching the levels after dexamethasone treatment (Figure 9B).
  • extramembrane vesicles have a more excellent inhibitory effect on IL-6, suggesting that they have better application prospects in the intervention treatment of cytokine storm suppression of new coronavirus pneumonia.
  • the above data indicate that the extramembrane vesicles of human adipose-derived mesenchymal progenitor cells can significantly inhibit the release of pro-inflammatory factors induced by LPS in macrophages, thereby inhibiting their pro-inflammatory response.
  • the exosomes were inhaled by aerosol, once a day for 5 days. After that, blood samples were collected for peripheral blood white blood cell and neutrophil counts and bacterial load measurement. Collect alveolar lavage fluid for neutrophil count, bacterial load measurement, and related pro-inflammatory factor level determination. In addition, the bacterial load of the lung tissue was measured, and the morphological observation of the lung tissue was performed by HE staining.
  • the enrollment criteria of this study mainly include 1) 18-75 years old, male or female, and I or my family members voluntarily join and sign the informed consent; 2) RT-PCR test positive or a clear diagnosis of new coronavirus pneumonia patients; 3 ) Meet the diagnostic criteria for severe and critically ill patients.
  • Exclusion criteria mainly include 1) related virus carriers or patients with severe allergies, 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 liver function Abnormal patients; 4) patients who are using ECMO or high-frequency oscillatory ventilation; 5) patients who are planning to give birth during pregnancy, lactation or within six months; 6) the investigator judges patients who cannot participate in the study or who fail to understand and implement the program.
  • some alternative cell therapies include embryonic stem cells (ESCs) treatment, induced pluripotent stem cells (iPSCs) treatment in vitro, and Mesenchymal stem cells (Mesenchymal stem cells).
  • ESCs embryonic stem cells
  • iPSCs induced pluripotent stem cells
  • Mesenchymal stem cells Mesenchymal stem cells
  • stromal/stem cells MSCs
  • lung epithelial progenitor cells Pulmonary epithelial progenitor cells, EpPCs
  • Endothelial progenitor cells Endothelial progenitor cells, EnPCs
  • MSCs are the most studied stem cell therapy. MSCs are a kind of pluripotent stem cells that can self-renew and proliferate. They can suppress the immune response in vitro and have the potential to differentiate into alveolar type 2 cells (AT2 cells). MSCs-mediated inflammation suppression and MSC-related lung repair and regeneration effects are likely to be the main treatment for lung diseases such as ARDS, pneumonia, asthma, chronic obstructive pulmonary disease (COPD) and pulmonary interstitial fibrosis (IPF). reason.
  • ARDS ARDS
  • COPD chronic obstructive pulmonary disease
  • IPF pulmonary interstitial fibrosis
  • ARDS is a serious clinical syndrome caused by the destruction of the alveolar epithelial barrier, accompanied by interstitial edema and inflammatory cell infiltration, leading to progressive acute respiratory failure.
  • synthetic corticosteroids, surfactants, inhaled nitric oxide, antioxidants, protease inhibitors, and various other anti-inflammatory treatments, such as simvastatin and ibuprofen many drugs have been used to treat ARDS, but No drug can significantly reduce the mortality of ARDS, and the mortality of ARDS is still maintained at 34%-44%.
  • ARDS is usually a complication of severe sepsis, especially after infection with Gram-negative bacteria. MSCs treatment can prevent the occurrence of ARDS in an animal model of sepsis induced by E. coli-derived lipopolysaccharide (LPS)[Curley G F,et al.Critical Care Med.2017,45(2):e202--e212.][Lee JW, et al. Stem Cell 2011, 29(6):913–919.].
  • LPS E. coli-derived lipopolysaccharide
  • MSCs In the ARDS mouse model induced by lipopolysaccharide, intravenous injection of MSCs can significantly improve alveolar injury and inflammation.
  • IL-10 interleukin-10
  • MSCs reduce the infiltration of neutrophils in the lungs and reduce the production of inflammatory factor TNF- ⁇ by immune cells infiltrating the lungs[Gupta N, et al. Journal of Immunology 2007,179,(3):1855–1863.][Mei SHJ,etal.PLoS Medicine,2007,4(9):e269.].
  • MSCs promote the regeneration of AT 2 cells, prevent endothelial cell apoptosis, and promote ARDS damage Repair of the lung epithelial barrier[Lee JW,et al. Proceedings of the National Academy of Sciences of the United States of America2009,106(38):16357-16362.][Hu S,et al.Stem Cell Research&Therapy2016,7( 1):66.].
  • KGF keratinocyte growth factor
  • VEGF vascular endothelial growth factor
  • HGF hepatocyte growth factor
  • MSCs extracellular vesicles
  • EVs extracellular vesicles
  • MSCs produce antibacterial proteins, which can directly inhibit the growth of bacteria in the inflamed lung.
  • Intratracheal injection of MSCs can significantly reduce lung injury and inflammation, and promote bacterial clearance in a lipocalcin-2-dependent manner, thereby increasing the survival rate of experimental animals with bacterial pneumonia.
  • the activation of TLR-4 in MSCs induced by LPS enhances the secretion of lipocalcin-2, and lipocalcin-2 can bind to the siderophore of bacteria, reduce iron absorption, and inhibit bacterial growth. Consistent with these findings are the recently reported findings: In an experimental model of bacterial pneumonia, Gupta et al. [Gupta N, et al. Thorax 2012, 67(6): 533–539] found mutations in TLR-4 in MSCs Significantly weakened their therapeutic effects. Therefore, it indicates a potential new cell-based treatment that may eliminate antibiotic-resistant Gram-negative strains in the lungs.
  • the research of the present invention shows that the mesenchymal stem cell has a significant advantage of extra-membrane vesicles.
  • Stem cells have great potential in the treatment of many diseases.
  • the therapeutic effect of stem cells is largely dependent on the paracrine cytokines and extracellular vesicles (EVs) or exosomes mediated.
  • EVs extracellular vesicles
  • EVs is a nano-sized membrane structure vesicle, which has the mediating function of cell-to-cell communication.
  • EVs derived from MSCs include cytokines, growth factors, signal lipids, mRNA, miRNA/siRNA and other substances.
  • MSC-EVs may represent a new cell-free treatment method. Compared with existing MSCs therapies, it has obvious advantages: 1) No risk of tumor formation in the body; 2) Lower 3) EVs can be derived from endogenous MSCs, or they can be obtained from the supernatant (conditioned medium) in the process of culturing allogeneic human MSCs through separation and purification, which can meet clinical-level large-scale production and purification.
  • the secondary yield is high, which is convenient for batch quality control and testing; 4)
  • the seed stem cells of specific proliferation generations that produce EVs can be modified in vitro to enrich more specific EVs for different therapeutic purposes; 5)
  • Single suspension state The diameter of MSC (18-40 ⁇ m) is 3-7 times the diameter of red blood cell (6 ⁇ m), and it is easy to deposit and adhere to form micro clots. After intravenous injection, there is a risk of clogging the capillaries.
  • EVs are nano-membrane structured vesicles in the diameter range of 50-500nm, which have good dispersibility in aqueous solution and are easy to be made into injection preparations; 6) EVs have good membrane structure stability and are more resistant to cryopreservation and freeze-thaw processes.
  • MSC-derived EVs Due to the membrane structure characteristics and nanoparticle characteristics of EVs, they can be actively swallowed and taken up by target tissue cells, thereby obtaining the biological information and/or active drugs carried by EVs in order to exert the direct signal of EVs to cells Conduction and targeted drug delivery functions. 8) EVs can be loaded with certain active drugs and become a natural drug delivery system. 9) Multiple research groups have reported the therapeutic potential of MSC-derived EVs. MSCs-EVs may be used as biomarkers and therapeutic agents for ARDS in the future. MSCs-EVs also seem to play a key role in the recovery process of ARDS.
  • MSC-derived microvesicles have therapeutic advantages through airway or intravenous administration.
  • Research by Zhu et al. showed that in the lung injury induced by E. coli endotoxin, intratracheal infusion of MVs derived from MSCs can reduce the extracellular water content of lung tissue, reduce pulmonary edema, and reduce the permeability of alveolar membranes to protein.
  • MSC-MVs also reduced the influx of neutrophils and reduced the level of macrophage inflammatory protein-2 in the alveolar lavage fluid (BAL).
  • MSCs regulate macrophages in ALI through EVs-mediated mitochondrial transfer.
  • MSC-MVs contain a large amount of Angiopoietin-1, and the immunomodulatory properties of MSCs on macrophages are partly mediated by the transfer of Angiopoietin-1 mRNA to macrophages.
  • HGF hepatocyte growth factor
  • MSCs exosomes reduce endothelial cell apoptosis, increase IL-10 production and reduce IL-6 production, and partially improve the LPS-induced acute lung injury model in mice. Pulmonary microvascular permeability.
  • MSCs-MVs can restore the integrity of the alveolar membrane tight junctions, and can also reduce the permeability enhancement effect of IL-1 ⁇ , TNF- ⁇ and IFN- ⁇ on human lung microvascular endothelial cells.
  • Anti-CD44 and Angiopoietin-1 siRNA pretreatment can eliminate this therapeutic effect of MSCs-MVs, indicating that the transfer of CD-44 and Angiopoietin-1 mRNA is involved in the repair and treatment mechanism of MSCs-MVs.
  • Pretreatment of MSCs can be used to enrich the expression of MVs subgroups, thereby increasing their therapeutic potential.
  • Invention Item 1 A pharmaceutical preparation containing an active substance derived from human cells.
  • the composition of the preparation has good stability and high dispersion, and is easily absorbed by the body except the skin. It is suitable for preventing and treating various causes Inflammation and injury.
  • Invention Item 2 The human cell-derived active substance of Invention Item 1, which includes, but is not limited to, extracellular vesicles and soluble active cytokines produced by stem cells, progenitor cells and immune cells derived from human tissues, bone marrow and blood .
  • Invention Item 3 The pharmaceutical preparation according to Invention Item 1, which includes, but is not limited to, aerosol inhalation liquid, eye drops, and nasal drops.
  • Invention Item 4 The pharmaceutical preparation described in Invention Item 1 has good stability, including but not limited to storage under normal temperature conditions, storage below -20°C, storage under low temperature conditions below -70°C, and low temperature conditions below -135°C When stored and stored at -196°C, the stability of the active substance is significantly better than the storage time of the preparation containing living cells under the same conditions.
  • Invention Item 5 The pharmaceutical preparation of Invention Item 1 has a high degree of dispersion, wherein it is colorless and transparent after standing for 6-24 hours under the condition of 0-25°C, and there is no visible flocs and sediments.
  • Invention Item 6 The pharmaceutical preparation described in Invention Item 1 is easy to use on other parts of the human body except skin, where these parts include, but are not limited to, eyeball conjunctiva, eyelid conjunctiva, retina, oral cavity, nasal cavity, upper respiratory tract, lower respiratory tract, stomach Intestinal tract, vascular endothelial cells, alveolar type 1 epithelial cells, alveolar type 2 epithelial cells, monocytes, macrophages, neutrophils, dendritic cells, antigen presenting cells, T lymphocytes, fibroblasts, central nervous system Cells, peripheral nerve cells and their peripheral nerve fibers.
  • eyeball conjunctiva eyelid conjunctiva
  • retina The pharmaceutical preparation described in Invention Item 1 is easy to use on other parts of the human body except skin, where these parts include, but are not limited to, eyeball conjunctiva, eyelid conjunctiva, retina, oral cavity, nasal cavity, upper respiratory tract, lower respiratory tract, stomach Intestinal tract, vascular
  • Invention Item 7 The pharmaceutical preparation described in Invention Item 1 is suitable for the prevention and treatment of inflammation and injury caused by various reasons, including but not limited to viral infectious inflammation, bacterial infectious inflammation, fungal infectious inflammation, and self Immunoreactive inflammation, ischemic injury, chemical injury, physical injury.
  • Invention Item 8 The human tissue, bone marrow, and blood-derived stem cells, progenitor cells, and immune cells described in Invention Item 2, including but not limited to mesenchymal stem cells derived from human adipose tissue, and alveolar epithelial progenitors derived from human lung tissue Cells, mesenchymal stem cells derived from human umbilical cord Wharton’s glue, mesenchymal stem cells derived from human endometrial tissue, stem cells derived from human placental tissue, epithelial cells derived from human placental amniotic membrane, mononuclear cells in human blood, Hematopoietic stem cells in human bone marrow tissue, mesenchymal stem cells in human bone marrow tissue, mesenchymal stem cells derived from human periosteum, keratinocytes derived from human skin tissue, dendritic cells derived from human blood tissue, and those derived from human blood tissue CD4-positive T lymphocytes, platelets
  • Invention Item 9 The human cell-derived extracellular vesicles according to Invention Item 2, which include, but are not limited to, nanovesicles surrounded by a lipid bilayer membrane structure, with a diameter between 30-1000 nanometers, It contains a wealth of different types of microribonucleic acid (miRNA), small ribonucleic acid (sRNA), non-coding DNA fragments, transport ribonucleic acid t-RNA, soluble cytokines, growth factors, and other proteins with specific functions.
  • miRNA microribonucleic acid
  • sRNA small ribonucleic acid
  • non-coding DNA fragments transport ribonucleic acid t-RNA
  • soluble cytokines growth factors, and other proteins with specific functions.
  • Invention Item 10 The soluble active cytokine derived from human cells according to Invention Item 2, which includes, but is not limited to, soluble free lipoprotein molecules, glycoprotein molecules, biosignal molecules, tissue growth factor B (TGF ⁇ ), Stem cell growth factor (HGF) vascular endothelial cell growth factor (VEGF), epidermal cell growth factor (KGF), interleukin-10 (IL-10), interleukin-1 receptor antagonist (IL-1 ⁇ A).
  • TGF ⁇ tissue growth factor B
  • HGF Stem cell growth factor
  • VEGF vascular endothelial cell growth factor
  • KGF epidermal cell growth factor
  • IL-10 interleukin-10
  • IL-1 ⁇ A interleukin-1 receptor antagonist
  • Invention Item 11 The soluble active cytokine derived from human cells described in Invention Item 2 and Invention Item 10, including but not limited to paracrine produced by human cells cultured in a natural state, and modifying human cells with specific genes Overexpressed.
  • Invention Item 12 The extracellular vesicles of Invention Item 2 and Invention Item 9, wherein the membrane protein markers CD9, CD63, CD81 and TSG101 are specifically expressed, but CANX is not expressed.
  • the diameter of extracellular vesicles is preferably 50-500 nm.
  • Invention Item 13 The human tissue, bone marrow, and blood-derived stem cells, progenitor cells, and immune cells described in Invention Item 2 and Invention Item 8, wherein these cells can be obtained by direct isolation and purification from human tissue, or these cells can be obtained from humans After the tissue is directly separated and purified, it undergoes minimal manipulation in the GMP laboratory to make these cells proliferate, or these cells undergo specific gene modification, specific gene editing, specific gene transduction, specific microribonucleic acid miRNA in the GMP laboratory The result of introduction, or the pretreatment of these cells under special culture conditions in a GMP laboratory.
  • Invention Item 14 The human tissue, bone marrow, and blood-derived stem cells, progenitor cells, and immune cells according to Invention Item 2 and Invention Item 8, wherein these cells are preferably adipose-derived mesenchymal stem cells or placental amniotic membrane-derived mesenchyme Cytoplasmic stem cells.
  • Inventive item 15 Inventive item 2, Inventive item 8, Inventive item 13, Inventive item 14, the stem cells, progenitor cells and immune cells derived from human tissues, bone marrow and blood described in Invention Item 14, more preferably fat-derived mesenchymal stem ( Progenitor) cells, mesenchymal stem cells derived from placental amniotic membrane.
  • Progenitor fat-derived mesenchymal stem
  • Invention Item 16 The adipose-derived stem (progenitor) cells described in Invention Item 15 are further preferably adipose-derived stem (progenitor) cells, wherein those that have undergone ethical review, have signed an informed consent, and have undergone strict laboratory inspections are in line with the establishment of adipose-derived stem cell products.
  • Standard male or female healthy volunteers have undergone abdominal wall puncture liposuction or abdominal wall surgery to cut fat, preserved in a patent-protected cell preservation solution, and transported to a qualified GMP laboratory at low temperature. After patent-protected separation, purification, Working bank cells (intermediate products) of proliferated adipose-derived mesenchymal progenitor cell products.
  • Invention Item 17 The further preferred human adipose tissue-derived mesenchymal progenitor cells described in Invention Item 15 and Invention Item 16, wherein the specific human adipose tissue-derived mesenchymal stem (progenitor) cells are in GMP
  • the production laboratory meets the negative standards of various pathogenic tests, and meets the specific surface markers such as CD73 positive, CD90 positive, CD105 positive cell percentage is about 98%, CD34 positive, CD45 positive and HLA -P1-P6 generation adipose-derived mesenchymal stem (progenitor) cells that have a percentage of DR-positive cells less than 2% and meet the criteria for antibiotic residues and serum and serum substitute residual detection standards, more preferably P3-P4 generation adipose-derived mesenchymal stem (progenitor) cells Stem (progenitor) cells.
  • Invention Item 18 The further preferred adipose tissue-derived mesenchymal stem (progenitor) cells described in Invention Item 15-17 are stored under deep low temperature conditions of -196°C to -80°C for 0-36 months, and more preferably -196°C to -135°C, stored for 0-24 months.
  • Invention Item 19 The human cell-derived extracellular vesicles and soluble active cytokines described in Invention Item 2 and Invention Items 9-12, wherein the production process is:
  • Invention Item 20 The differential centrifugation combined with polyethylene glycol PEG precipitation described in the production process (2) of Invention Item 19, wherein PEG3000-PEG9000 is used, 8%-30% PEG is configured with PBS, and After filtering with a 0.22 ⁇ m filter, add it to the treated conditioned medium at a volume ratio of 1:1, incubate overnight at 4°C, centrifuge at 3,000-5000g for 45-60 minutes at 4°C, and discard the supernatant.
  • Invention Item 21 The extracellular vesicles obtained by the production process of Invention Item 19 and Invention Item 20 are characterized by the biomarkers CD9 positive, CD63 positive, CD81 positive, TSG101 positive and CANX negative.
  • Invention Item 22 The production process described in Invention Items 19-21, in which, under GMP laboratory conditions, it can be scaled up to the clinical-level production scale of cell products, and its outstanding advantages are: 1 production operation unit can separate 2 cell factories The 800-1200ml conditioned medium obtained can isolate 2-5 ⁇ 10 11 extracellular vesicles. Calculated according to the total dose of 2-5 ⁇ 10 9 extracellular vesicles used locally for a patient, one production operation unit and one batch of production can meet the local consumption of 100-250 patients. With 100 production operation units, a batch of production can reach a total of 10,000-25,000 people, and it will take 5-6 days.
  • Invention Item 23 The production process according to Invention Item 19-22, wherein the separated extracellular vesicles, soluble active cytokines and PEG of specific molecular weight are cross-linked to form hydrophilic particles less than 3 microns at -196°C
  • the stability under freezing conditions at -20°C and the dispersibility in an aqueous solution at room temperature are better than those of the prior art.
  • Invention Item 24 The extracellular vesicles, soluble active cytokines, and polyethylene glycol PEG cross-linked hydrophilic particles according to Invention Item 23, including but not limited to those in isotonic sodium chloride solution, low molecular transparent From the acid solution, artificial tears, and gel solutions, it is prepared into aerosolized inhalation liquid, eye drops, and nasal drops for external use.
  • the uptake of the extracellular vesicles obtained by the process of the present invention by local mucosal cells is not affected by PEG.
  • Invention Item 25 The pharmaceutical preparations containing human extracellular membrane vesicles and soluble active cytokines described in Invention Item 1 and Invention Items 23-24 are preferably prepared as aerosol inhalants, which are suitable for the treatment of infectious lung injury.
  • Invention Item 26 The pharmaceutical preparations of Invention Item 1 and Invention Item 25, wherein adipose tissue-derived stem (progenitor) cells and placental amniotic membrane-derived mesenchymal stem cell-derived extramembrane vesicles and soluble cytokines are preferred and isotonic
  • the aerosol inhalation liquid prepared by sodium chloride aqueous solution is suitable for viruses, including but not limited to influenza virus, SARS-coronavirus, SARS-coronavirus 2, MERS-coronavirus infection, and aerosol inhalation treatment of acute lung injury caused by infection, in order to prevent Acute lung injury releases inflammatory factors, reduces the infiltration of high protein fluid in the alveoli and alveolar epithelial cell damage, significantly improves the rescue success rate of patients with acute lung injury, and improves the lung function and quality of life of surviving patients.
  • viruses including but not limited to influenza virus, SARS-coronavirus, SARS-coronavirus 2, MERS-cor

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Abstract

一种可用于治疗ARDS等炎症或损伤相关的疾病的药物组合物，它含有（a）人的体细胞衍生的活性物质，所述活性成分为人的体细胞产生的细胞膜外囊泡；和（b）药学上可接受的载体，所述体细胞选自人体组织、骨髓和/或血液来源的干细胞等，所述药物组合物的剂型选自雾化吸入剂、滴眼剂、滴鼻剂，所述药物组合物具有优于活细胞制剂的保存稳定性和高分散性。一种制备细胞膜外囊泡的方法，包括培养人的体细胞、从培养体系中去除细胞、将培养液与聚乙二醇混合形成经PEG修饰的细胞膜外囊泡、离心、重悬等步骤。所述细胞膜外囊泡制剂可用于制备预防和/或治疗炎症、或损伤的药物。

Description

含人体细胞衍生的细胞膜外囊泡的雾化吸入制剂、制法及其应用 技术领域
本发明涉及生物医药领域，具体地涉及含人体细胞衍生的细胞膜外囊泡的雾化吸入制剂、制法及其应用。
背景技术
急性呼吸窘迫综合征(acute respiratory distress syndrome,ARDS)是危重病人呼吸衰竭的常见原因，其定义为急性发作的非心源性肺水肿、低氧血症和需要机械通气。
ARDS最常见于肺炎、败血症、胃内容物误吸或严重创伤，约占全世界重症监护病房患者的10％。尽管在过去的几十年里取得了一些进步，但在大多数研究中，死亡率仍然高达30-40％。在美国每年总计约20万例ARDS，医院死亡率高达38.5％，而且在过去的几十年里，这种状况并没有明显的改善。
急性肺损伤(acute lung injury,ALI)和ARDS具有性质相同的病理改变，最常见的是弥漫性肺泡损伤。ALI/ARDS的病理基础是由多种炎症细胞(巨噬细胞、嗜中性粒细胞和淋巴细胞等)介导的肺局部炎症反应和炎症反应失控所致的肺泡上皮和肺泡毛细血管内皮损伤。其主要病理特征为由肺微血管通透性增高而导致的肺泡渗出液中富含蛋白质的肺水肿及透明膜形成，可伴有肺间质纤维化。病理生理改变以肺顺应性降低，肺内分流增加及通气血流比例失衡为主。临床表现为呼吸频数和呼吸窘迫、顽固性低氧血症，胸部X线显示双肺弥漫性浸润影，后期常并发多器官功能衰竭。
ARDS的诊断是基于专家共识的临床诊断标准。患者管理的重点是实施肺保护通气策略。目前还没有确定具体的药物疗法。ARDS存活患者的长期预后越来越被认为是重要的研究目标，因为许多ARDS患者存活下来后，器官功能和/或认知心理等后遗症仍在继续。未来的研究方向包括促进ARDS的早期识别，在临床研究中增加预后和/或预测功能，以识别可能具有治疗效果的亚群，以及继续努力了解肺损伤的基本机制。
现有的用于治疗急性呼吸窘迫综合征的方法旨在调节炎症或炎症级联效应的治疗方法，已被用于治疗ARDS的研究。然而，目前抗炎治疗药物如皮质类固醇、中性粒细胞弹性蛋白酶抑制剂、粒细胞集落刺激因子、他汀类药物和ω-3脂肪酸，以及针对改善肺呼吸力学的表面活性剂如吸入β受体激动剂和一氧化氮等，都未能显示降低死亡率的收益。只有减少机械通气过程中压力性肺损伤(气压性创伤)的支持性治疗，如使用神经肌肉阻滞剂的肺保护性通气或俯卧位通气，才显示出死亡率的改善，因此这些治疗仍然是ARDS治疗的主要手段。截止目前，ARDS现有的治疗方法，既不能降低死亡率，也不能降低存活患者的肺纤维化等器官功能损害以及神经精神等方面的后遗症。
2019年底和2020年，在中国和世界各地爆发新型冠状病毒(SARS-CoV-2)感染性肺炎，引起重症肺炎和急性呼吸窘迫综合征(ARDS)等。针对SARS-CoV-2等冠状病毒导致的重症肺炎和ARDS，目前尚无特效的疫苗和抗病毒药物治疗。这些感染性疾病严重影响了人类的生命健康，研发效果好的治疗药物迫在眉睫。针对SARS-CoV-2等冠状病毒引起的肺炎和ARDS等疾病开发出低毒高效的药物，以满足国内外患者的临床需求，具有重大的社会意义。
综上所述，本领域迫切需要开发高效、低毒、安全和可大规模生产的治疗ARDS等疾病的药物。
发明内容
本发明的目的是提供一种高效、低毒、安全和可大规模生产的治疗ARDS等疾病的药物。
在本发明的第一方面，提供了一种药物组合物，所述的药物组合物含有(a)人的体细胞衍生的活性物质,所述的活性物质为人的体细胞所产生的细胞膜外囊泡；和(b)药学上可接受的载体。
在另一优选例中，所述的药物组合物是无细胞的药物组合物。
在另一优选例中，所述的“无细胞”指所述的药物组合物不含有活细胞和死细胞。
在另一优选例中，所述的体细胞选自下组：人体组织、骨髓和/或血液来源的干细胞、祖细胞、免疫细胞、或其组合。
在另一优选例中，所述的药物组合物的剂型选自下组：液态剂型、固态剂型(如冻干剂型)。
在另一优选例中，所述的药物组合物的剂型选自下组：雾化吸入剂、滴眼液、滴鼻液。
在另一优选例中，所述的药物组合物具有以下特性：
(P1)优于活细胞制剂的保存稳定性；优选地，所述的保存包括在常温和/或低温下(如-196℃至25℃，较佳地-100℃至0℃)进行保存；所述的“优于活细胞制剂的保存稳定性”指所述的药物组合物的稳定性优于相同条件下含活细胞制剂的稳定性；和
(P2)高分散性；较佳地，当所述药物组合物为含水的液态制剂时(如以生理盐水配制的溶液时)，在0-25℃放置6-24小时，呈现无色透明，且无肉眼可见的絮状物和沉淀物。
在另一优选例中，所述的药物组合物被施用于除了皮肤之外的部位。
在另一优选例中，所述的药物组合物被施用于选自下组的部位：眼球结膜、眼睑结膜、视网膜、口腔、鼻腔、上呼吸道、下呼吸道、胃肠道、肺部、或其组合。
在另一优选例中，所述的药物组合物被施用于选自下组的细胞：血管内皮细胞、 肺泡1型上皮细胞、肺泡2型上皮细胞、单核巨噬细胞、中性粒细胞、树突状细胞、抗原递呈细胞、T淋巴细胞、纤维母细胞、中枢神经细胞、周围神经细胞及其末梢神经纤维、或其组合。
在另一优选例中，所述的干(祖)细胞选自下组：人体脂肪组织来源的间充质干细胞、人体肺组织来源的肺泡上皮祖细胞、人体脐带华通氏胶来源的间充质干细胞、人体子宫内膜组织来源的间充质干细胞、人体胎盘组织来源的干细胞、人体胎盘羊膜来源的上皮细胞、人体血液中的单个核细胞、人体骨髓组织中的造血干细胞、人体骨髓组织中的间充质干细胞、人体骨膜来源的间充质干细胞、人体皮肤组织来源的角质细胞、人体血液组织来源的树突状细胞、人体血液组织来源的CD4阳性T淋巴细胞、人体血液组织来源的血小板、或其组合。
在另一优选例中，所述的细胞膜外囊泡包括由脂质双分子层膜结构包绕形成的纳米囊泡。
在另一优选例中，所述的纳米囊泡的直径在30-1000纳米之间。
在另一优选例中，所述的纳米囊泡内含有不同种类的、具有特定功能的微小核糖核酸(miRNA)、小核糖核酸(sRNA)、非编码的DNA片段、转运核糖核酸(t-RNA)、可溶性细胞因子、生长因子、和其他蛋白质。
在另一优选例中，所述的可溶性活性细胞因子选自下组：可溶性的游离的脂蛋白质分子、糖蛋白分子、生物信号分子、组织生长因子β(TGFβ)、肝细胞生长因子(HGF)、血管内皮细胞生长因子(VEGF)、表皮细胞生长因子(KGF)、白细胞介素-10(IL-10)、白细胞介素-1β受体拮抗剂(IL-1βA)、或其组合。
在另一优选例中，所述的可溶性活性细胞因子是所述的人的体细胞所产生的可溶性活性细胞因子。
在另一优选例中，所述的可溶性活性细胞因子包括自然状态下培养的人的体细胞旁分泌所产生的活性细胞因子、用外源基因修饰的人的体细胞所表达产生的活性细胞因子、或其组合。
在另一优选例中，所述的细胞膜外囊泡特异性表达以下蛋白：CD9、CD63、CD81和TSG101；不表达或基本不表达蛋白CANX。
在另一优选例中，所述的细胞膜外囊泡的直径优选为50-500nm。
在另一优选例中，所述的直径为平均直径。
在另一优选例中，用于生产所述细胞膜外囊泡的细胞包括以下来源的细胞：
(a)从人体组织直接分离纯化所获得的细胞；
(b)从人体组织直接分离纯化后，再经过GMP实验室最小程度的操作，使这些细胞增殖后所获得的细胞；
(c)在GMP实验室经过特定基因修饰、特定基因编辑、特定基因转导、特定微小核糖核酸miRNA的导入所获得的细胞；
(d)在GMP实验室经过特殊培养条件的预处理所获得的细胞。
在另一优选例中，所述的用于生产所述细胞膜外囊泡的细胞包括原代细胞、传代次数为1-10次的传代细胞。
在另一优选例中，所述的用于生产所述细胞膜外囊泡的细胞包括未经基因操作的细胞、经基因操作的细胞。
在另一优选例中，所述的基因操作包括基因编辑、基因导入、基因下调(knock-down)、基因敲除(knock-out)、或其组合。
在另一优选例中，所述的用于生产所述细胞膜外囊泡的细胞是经预处理的细胞。
在另一优选例中，所述的体细胞为脂肪来源的间充质干(祖)细胞、胎盘羊膜来源的间充质干细胞、或其组合。
在另一优选例中，所述的脂肪来源的间充质干(祖)细胞是通过以下方式获得的：对于经过伦理审查备案的、签署知情同意书的、经过严格实验室检查符合脂肪干(祖)细胞产品建库入组标准的男性或女性健康志愿者，经过腹壁皮肤穿刺抽脂、或腹壁手术切取脂肪，置于细胞保存液中保存的、低温运输至合格的GMP实验室，经过分离、纯化、增殖处理，所获得的脂肪间充质祖细胞，即工作库细胞(中间产品)。
在另一优选例中，所述的人体脂肪组织来源的间充质干(祖)细胞是在GMP生产实验室，按照中间产品生产工艺流程生产的、符合各种病原学检测阴性标准的、符合特定表面标志物如CD73阳性、CD90阳性、CD105阳性细胞百分率大约98％，CD34阳性/CD45阳性和HLA-DR阳性的细胞百分率小于2％的、符合抗生素残余和血清及血清替代物残余检测标准的、P1-P6代脂肪间充质干(祖)细胞。
在另一优选例中，所述的脂肪间充质干(祖)细胞为P3-P4代脂肪间充质干(祖)细胞。
在另一优选例中，所述的体细胞是在-196℃至-80℃(较佳地，-196℃至-135℃)深低温条件下储存0-36个月(较佳地0-24个月或0.5-24个月)的脂肪组织来源的间充质干(祖)细胞。
在另一优选例中，所述的人的体细胞衍生的细胞膜外囊泡及可溶性活性细胞因子，其生产工艺包括步骤：
(1)通过接种所述的人的体细胞于含5％EliteGro的αMEM基础培养基中，在HyperFlask培养瓶中培养，培养条件为37±1℃，5±0.5％CO 2,至细胞融合度达到80±10％，再使用特定的预处理培养基进行培养，培养条件为37±1℃，5±0.5％CO 2，培养36-72小时；
(2)收集细胞培养上清液，进行差速离心联合聚乙二醇(PEG)沉淀法分离。
在另一优选例中，所述的差速离心分离联合聚乙二醇PEG沉淀，包括步骤：使用PEG3000-PEG9000的PEG，用PBS配置为8％-30％的PEG，经过滤除菌(如用0.22μm的过滤器过滤)后，按照一定比例(如约1:1体积比)加入到处理后的条件培养基中，置 于4℃过夜孵育后。4℃，3,000-5000g离心30-60分钟，弃去上清。加入预冷的PBS重悬沉淀，4℃，100000-120000g超高速离心60-120分钟，弃去上清，加入适量等渗氯化钠溶液，重悬沉淀，从而获得人体细胞衍生的细胞膜外囊泡及可溶性活性细胞因子。
在另一优选例中，用所述生产工艺获得的细胞膜外囊泡，具有以下生物标志物特征：CD9阳性(即CD9 +)、CD63阳性、CD81阳性、TSG101阳性、和CANX阴性。
在另一优选例中，所述的生产工艺具有以下特性：在GMP实验室条件下，具有临床级细胞制品生产规模。
在另一优选例中，所述的临床级生产规模指1个生产操作单元可以分离2个细胞工厂所获得的800-1200ml条件培养基，可分离获得2-5×10 11个细胞膜外囊泡，按照一个病人2-5×10 9个细胞膜外囊泡在局部使用的总剂量计算，一个生产操作单元，一个批次生产量可以满足100-250个病人的局部使用量。或者，100个生产操作单元，一个批次生产量可达到10000-25000人使用总量，用时5-6天。
在另一优选例中，所述的生产工艺的特点是：分离获得的细胞膜外囊泡、可溶性活性细胞因子与特定分子量的PEG交联成特定粒径(如小于3微米)的亲水性粒子，从而具有在-196℃至-20℃冻存条件下的高稳定性，以及在常温水溶液中的高分散性。
在另一优选例中，所述的细胞膜外囊泡、可溶性活性细胞因子及聚乙二醇PEG交联的亲水粒子，分散于等渗氯化钠溶液、低分子透明质酸溶液、人工泪液、凝胶溶液中，从而制成雾化吸入液、滴眼液、滴鼻液、和外用凝胶制剂。
在另一优选例中，所述的药物组合物并施用于粘膜细胞，并且所述粘膜细胞可摄取细胞膜外囊泡。
在另一优选例中，本发明的药物组合物为雾化吸入剂。
在另一优选例中，本发明的药物组合物(尤其是雾化吸入剂)被用于治疗感染性肺损伤，较佳地用于病毒性肺损伤，更佳地用于冠状病毒(如SARS-CoV病毒、SARS-CoV-2病毒)感染引起的肺损伤。
在另一优选例中，所述的药物组合物是脂肪组织来源的祖细胞和胎盘羊膜来源的间充质干细胞衍生的膜外囊泡和可溶性细胞因子与等渗氯化钠溶液制备的雾化吸入液。
在另一优选例中，所述的药物组合物用于治疗病毒引起的疾病。
在另一优选例中，所述的病毒选自下组：流感病毒、SARS冠状病毒、SARS冠状病毒2、MERS冠状病毒感染。
在另一优选例中，所述的药物组合物(如雾化吸入液)用于治疗病毒性急性肺损伤，通过雾化吸入治疗，从而达到阻止急性肺损伤炎症因子的释放、减轻肺泡内高蛋白液的渗入和肺泡上皮细胞损伤、显著提高急性肺损伤患者的抢救成功率，以及改善存活患者肺功能和生活质量。
在本发明的第二方面，提供了一种制备细胞膜外囊泡的方法，包括步骤：
(S1)培养人的体细胞(如脂肪间充质干(祖)细胞)，使其达到预定的汇合度(如75-90％)；
(S2)在适合产生EV的条件下，继续培养所述的细胞一段时间T1；其中，所述的T1通常为24-72小时，较佳地30-60小时；
(S3)从培养体系中去除细胞，从而分离获得含细胞膜外囊泡的培养液，即为“条件培养液(conditioned medium)”；
(S4)将所述的条件培养液(conditioned medium)与聚乙二醇(PEG)进行混合，形成第一混合物，并放置一段时间T2，从而形成经PEG修饰的细胞膜外囊泡；其中，所述的T2通常为6-60小时，较佳地12-48小时；
(S5)对上一步骤的第一混合物进行离心，使得所述的经PEG修饰的细胞膜外囊泡沉淀，并弃去上清，获得经PEG修饰的细胞膜外囊泡沉淀；
(S6)对上一步骤获得的经PEG修饰的细胞膜外囊泡沉淀进行重悬，从而获得第一重悬混合物；
(S7)对第一重悬混合物进行离心，使所述的经PEG修饰的细胞膜外囊泡沉淀，并弃去上清，获得经PEG修饰的细胞膜外囊泡沉淀；
(S8)对上一步骤中获得的经PEG修饰的细胞膜外囊泡沉淀进行重悬，从而获得可医用的细胞膜外囊泡制剂(即PEG-EVs-factors制剂)。
在另一优选例中，所述方法还包括：
(S9)将可医用的细胞膜外囊泡制剂(或活性物质)与药学上可接受的载体混合，从而制得药物组合物。
在另一优选例中，所述方法还包括将所述的药物组合物制成雾化吸入制剂、注射剂、或冻干制剂。
在本发明的第三方面，提供了一种细胞膜外囊泡制剂，所述的细胞膜外囊泡制剂是用本发明第二方面所述的方法制备的。
在另一优选例中，所述的细胞膜外囊泡制剂包括由脂质双分子层膜结构包绕形成的纳米囊泡。
在另一优选例中，所述的纳米囊泡的直径在30-1000纳米之间。
在另一优选例中，所述的纳米囊泡内含有不同种类的、具有特定功能的微小核糖核酸(miRNA)、小核糖核酸(sRNA)、非编码的DNA片段、转运核糖核酸(t-RNA)、可溶性细胞因子、生长因子、和其他蛋白质。
在另一优选例中，所述的可溶性活性细胞因子选自下组：可溶性的游离的脂蛋白质分子、糖蛋白分子、生物信号分子、组织生长因子β(TGFβ)、肝细胞生长因子(HGF)、 血管内皮细胞生长因子(VEGF)、表皮细胞生长因子(KGF)、白细胞介素-10(IL-10)、白细胞介素-1β受体拮抗剂(IL-1βA)、或其组合。
在另一优选例中，所述的可溶性活性细胞因子是所述的人的体细胞所产生的可溶性活性细胞因子。
在另一优选例中，所述的可溶性活性细胞因子包括自然状态下培养的人的体细胞旁分泌所产生的活性细胞因子、用外源基因修饰的人的体细胞所表达产生的活性细胞因子、或其组合。
在另一优选例中，所述的细胞膜外囊泡特异性表达以下蛋白：CD9、CD63、CD81和TSG101；不表达或基本不表达蛋白CANX。
在本发明的第四方面，提供了一种本发明第一方面所述的药物组合物或第三方面所述的细胞膜外囊泡制剂的用途，它们被用于制备预防和/或治疗炎症、或损伤的药物。
在另一优选例中，所述的炎症选自下组：病毒感染性炎症、细菌感染性炎症、真菌感染性炎症、自身免疫反应性炎症、或其组合；
所述的损伤选自下组：缺血性损伤、缺氧性损伤、化学性损伤、物理性损伤、或其组合。
在另一优选例中，所述的药物用于治疗病毒引起的疾病。
在另一优选例中，所述的病毒选自下组：流感病毒、SARS冠状病毒、SARS冠状病毒2、MERS冠状病毒感染。
在本发明的第五方面，提供了一种预防和/或治疗炎症、或损伤的的方法，包括步骤：给需要的对象施用本发明第一方面所述的药物组合物，或第三方面所述的细胞膜外囊泡制剂。
在另一优选例中，所述的炎症选自下组：病毒感染性炎症、细菌感染性炎症、真菌感染性炎症、自身免疫反应性炎症、或其组合。
在另一优选例中，所述的损伤选自下组：缺血性损伤、缺氧性损伤、化学性损伤、物理性损伤、或其组合。
在另一优选例中，所述的药物用于治疗急性呼吸窘迫综合征。
在另一优选例中，所述的药物为雾化吸入液。
在另一优选例中，所述的药物用于治疗病毒性急性肺损伤。
在另一优选例中，所述的药物通过雾化吸入治疗，从而达到阻止急性肺损伤炎症因子的释放、减轻肺泡内高蛋白液的渗入和肺泡上皮细胞损伤。
在另一优选例中，所述的对象是人。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了Western印迹法和NTA检测PEG沉淀法和超速离心法收集的细胞膜外囊泡的产量。其中，图(1A)：M为Marker；1～4分别为8％PEG 6000、12％PEG 6000、16％PEG 6000、20％PEG 6000分离出的细胞膜外囊泡的CD63表达量(同蛋白量上样)；图(1B)：M为Marker；1为haMSCs；2和4为超速离心分离的细胞膜外囊泡的蛋白表达情况；3和5为12％PEG 6000分离出的细胞膜外囊泡的蛋白表达情况；图(1C)：NTA检测PEG 6000和超速离心分离出的细胞膜外囊泡粒子浓度；图(1D)：NTA检测PEG6000和超速离心分离出的细胞膜外囊泡粒径大小；*p<0.05。
图2显示了成纤维细胞摄取细胞膜外囊泡。其中，蓝色：Hochest染色的细胞核；绿色：PKH-67标记的细胞膜外囊泡；标尺：100μm。
图3显示了在4℃、-20℃和-80℃条件下贮存9周的细胞膜外囊泡数量。
图4显示了在4℃、-20℃和-80℃条件下贮存9周的细胞膜外囊泡的特异性标志物CD81的表达情况。
图5显示了常温复融半小时后,透射电镜下的细胞膜外囊泡状态。其中，图5A为-80℃贮存1周的细胞膜外囊泡在透射电镜的状态，图5B为复融常温半小时后的细胞膜外囊泡在透射电镜的状态。
图6显示了常温复融半小时后细胞膜外囊泡的粒径浓度和大小。
图7显示了人源脂肪间充质祖细胞细胞膜外囊泡抑制LPS诱导巨噬细胞活化。其中，(A)促炎症型巨噬细胞形态学观察；(B)促炎症型巨噬细胞形态学统计分析。Control为对照组，LPS为单独使用脂多糖处理巨噬细胞组，LPS+Dex为脂多糖和地塞米松处理组，LPS+haMPCs-Exo为脂多糖和脂肪间充质祖细胞膜外囊泡处理组，红色箭头表示活化的巨噬细胞(促炎症型巨噬细胞)；*P<0.05，标尺为50μm。
图8显示了人源脂肪间充质祖细胞膜外囊泡抑制LPS诱导巨噬细胞促炎症因子基因表达。其中，(A)、(B)和(C)分别显示了实时荧光定量PCR检测巨噬细胞炎症因子TNF-α、IL-1β和IL-6的基因表达水平。Control为对照组，LPS为单独使用脂多糖处理巨噬细胞组，LPS+Dex为脂多糖和地塞米松处理组，LPS+haMPCs-Exo为脂多糖和脂肪间充质祖细胞膜外囊泡处理组；***P<0.001。
图9显示了人源脂肪间充质祖细胞膜外囊泡抑制LPS诱导巨噬细胞促炎症因子释放。其中，(A)LPS诱导巨噬细胞并经人源脂肪间充质祖细胞膜外囊泡处理流程示意图，(B)ELISA检测巨噬细胞释放的炎症因子IL-6和TNF-α蛋白水平。Control为对照组，LPS为单独使用脂多糖处理巨噬细胞组，LPS+Dex为脂多糖和地塞米松处理 组，LPS+haMPCs-Exo为脂多糖和脂肪间充质祖细胞膜外囊泡处理组；**P<0.01，***P<0.001。
图10为细胞膜外囊泡、可溶性细胞因子与PEG交联形成亲水性粒子的示意图。
图11显示了本发明一种示意性的工艺路线(从步骤S4到S8)。
具体实施方式
本发明人经过广泛而深入的研究，通过大量筛选和研究，首次意外地开发了一类可有效治疗ARDS的无细胞的生物医药制剂，该生物医药制剂含有人体细胞来源的细胞膜外囊泡，具有特定的粒径并且经特定PEG修饰，因此不仅可以稳定地长期保存，而且在医用溶剂(如生理盐水等)中具有极其优异的分散性，特别适合通过雾化吸入方式等方式直接施用于患者的肺部(尤其是下呼吸道)等部位，从而快速、高效地治疗或缓解肺炎、ARDS等疾病的症状，显著降低冠状病毒等因素所导致的感染症状和死亡率。在此基础上完成了本发明。
冠状病毒
冠状病毒(Coronavirus，CoV)属于套式病毒目(Nidovirales)冠状病毒科(Coronaviridae)，是一种有包膜的正链RNA病毒，其亚科包含α、β、δ及γ四属。
目前已知的感染人的冠状病毒中，HCoV-229E和HCoV-NL63属于α属冠状病毒，HCoV-OC43、SARS-CoV、HCoV-HKU1、MERS-CoV和SARS-CoV-2均为β属冠状病毒。
2003年和2012年分别爆发的高致病性冠状病毒“非典”SARS-CoV和“中东呼吸综合征”MERS-CoV均属于β属冠状病毒。2019年年底爆发的新型冠状病毒(SARS-CoV-2)与SARS-CoV有约80％相似性、与MERS-CoV有40％的相似性，也属于β属冠状病毒。
细胞膜外囊泡
1.间充质干细胞膜外囊泡的生物学特征
细胞膜外囊泡(extracellular vesicles,EVs)有三个主要类型，分别为外泌体(Exosomes)、微囊泡(Microvesicles)和凋亡小体(Apoptotic bodies)。所有这三个主要类型的EVs都被脂质双分子层包裹，其直径范围在30-2000nm。
外泌体一词指的是由胞内体衍生的直径在50-100nm的EVs亚类，它们是包括间充质干细胞(mesenchymal stem cells,MSCs)在内的多种细胞类型旁分泌物的主要组成部分。
MSCs外泌体(Exosomes)是MSCs衍生的EVs中的一类直径在50-100nm范围内的、具有完整脂质双分子层膜结构的EVs。
外泌体是一种运载丰富货物的载体，其功能主要是通过不断转运微小核糖核酸 (miRNAs)和蛋白来发挥的。在MSCs来源的外泌体中，已经鉴定出150多个miRNAs和850多个独特的蛋白质，通过不同的途径，改变靶细胞的各种活性。MSC外泌体参与了机体发育、表观遗传调控、免疫调节(miR-155和miR-146)、肿瘤发生和肿瘤进展(miR-23b、miR-451、miR-223、miR-24、miR-125b、miR-31、miR-214和miR-122)等生理和病理过程。根据ExoCarta的资料，已经从MSCs外泌体中收集了900多种蛋白质。一些研究表明，MSC外泌体载有一些细胞因子和生长因子,如TGFβ1、白细胞介素-6(IL-6)、IL-10和肝细胞生长因子(HGF)等，这些因子已被证实有助于免疫调节。血管内皮生长因子(VEGF)、细胞外基质金属蛋白酶诱导剂(extracellular matrix metalloproteinase inducer,EMMPRIN)和MMP-9在MSC外泌体中均有报道，这三种蛋白在刺激血管生成中发挥重要作用，可能是外泌体组织修复作用的基础。
2.间充质干细胞膜外囊泡在动物体内的生物分布
在不同的动物模型中，已经采用了几种体内示踪策略来确定EVs或外泌体在全身给药后的生物分布。近红外(Near-infrared，NIR)染料因其信噪比高，是体内应用的理想染料。标记超顺磁性氧化铁纳米颗粒的EVs具有高分辨率和灵敏的磁共振分析功能，为深部器官的准确检测提供了依据。
在脑出血大鼠模型中，经静脉注射后，DiI标记的MSC外泌体可以被显示到达脑、肝、肺和脾。在急性肾损伤(AKI)小鼠模型中，与健康对照组相比，DID标记的EVs在AKI小鼠肾脏中特异性积聚，预示着外泌体似乎能够归巢到损伤部位。
与静脉注射相比，经鼻给药可使损伤部位的脑外泌体更好地积聚。EVs经系统给药的生物分布是一个动态过程:给药后约30分钟内，EVs在肝脏、脾脏和肺中的快速分布阶段，然后通过肝脏和肾脏处理进入消除阶段，在给药后1至6小时内清除EVs。
3.间充质炎细胞膜外囊泡的免疫调节和抗炎作用
研究表明，外泌体通过携带和递呈功能性MHC肽复合物，在调节肿瘤特异性T细胞活化方面起着关键作用。
骨髓来源的MSCs释放的外泌体通过抑制CD4阳性T细胞的活化和浸润，减少促炎细胞因子的产生，促进表达IL-10的Treg的生成，抑制Th17细胞，可有效改善小鼠慢性移植物抗宿主病(cGVHD)。
人多能基质细胞来源的EVs在1型糖尿病(T1D)和实验性自身免疫性葡萄膜视网膜炎(EAU)模型中具有抑制自身免疫的作用。EVs可通过抑制抗原提呈细胞(APC)的活化，抑制Th1和抑制Th17细胞的发育，增加免疫抑制因子IL-10的表达。
人骨髓MSCs外泌体可以促进调节性T细胞亚群增殖和通过上调外周血单核细胞(PBMCs)表达细胞因子IL-10和TGF-β1,使哮喘病人的免疫抑制能力增强。
人脐带MCSs来源的外泌体中miR-181c通过下调TLR4信号通路在烧伤大鼠炎症 模型中发挥抗炎作用。
单次肾内注射猪自体脂肪MSCs-EVs可以使肾动脉狭窄猪模型的肾静脉中多种促炎细胞因子(TNF-α,IL-6,IL-1-β)的水平降低，而抑制炎症因子IL-10水平升高，并伴有巨噬细胞从促炎细胞向修复性巨噬细胞的迁移，再次证明了EVs的免疫调节潜能。
已有不少研究报道MSCs在肝纤维化/肝硬化动物模型和急性肝损伤中的应用，最终可以改善患者的病情进展。脂肪MSCs外泌体可以显著降低刀豆蛋白A(Con A)诱导的C57BL/6小鼠肝炎模型的血清丙氨酸转氨酶和天冬氨酸转氨酶水平的升高，减轻肝脏炎症，降低肝炎模型小鼠血清中促炎细胞因子(TNF-α、IFN-γ、IL-6、IL-18、IL-1β的水平，抑制了模型小鼠肝脏中炎性小体(inflammasome)的激活。
脓毒症是一种机体对抗微生物感染所引起的全身性炎症反应。尽管有先进的抗生素的应用，但是重症监护病房的脓毒血症死亡率仍然居高不下，于是研究者们将MSCs的治疗目标锁定在此类全身炎症性疾病。近年来，MSC-EVs在盲肠结扎致脓毒血症的动物模型中的疗效被广泛研究。在脓毒症大鼠模型中，静脉给予脂肪MSCs-EVs的治疗减轻了全身性炎症反应、器官损伤和随后的致死性。另一项治疗脓毒症的研究中，使用IL-1β预处理的MSCs产生的EVs治疗脓毒血症的效果比无预处理的MSCs来源的EVs有显著的提高。
研究还表，从诱导的MSCs得到的EVs能有效地极化巨噬细胞，使其分化为M2型，M2型是巨噬细胞的抗炎表型。miR-146a作为一种抗炎作用的miRNA,在IL-1β预处理MSCs及其EVs中的含量都显著增加。将EVs中包装的miR-146a转入巨噬细胞，可以使其极化为M2型[Casado JG,et al.Frontiers in Veterinary Science.2017；4:39]
4.基因修饰MSCs可增加其细胞膜外囊泡的疗效
为了改善人骨髓MSCs向肺的迁移和增强MSCs的治疗潜力，一种含有AT2R DNA的构建物，通过慢病毒介导的基因转染，使其在骨髓MSCs中获得高而稳定的长期表达。体外检测了AT2R-MSCs(AT2R-MSCs)的迁移。随后，在脂多糖(LPS)诱导的急性肺损伤体内模型中研究AT2R-MSCs的潜在治疗效率，并评估了MSCs向损伤肺组织的迁移。与此同时，使用AT2R基因敲除的MSCs(MSC-ShAT2R)，以确定AT2R在MSCs体内迁移能力中的作用以及对ARDS的治疗作用。
MSCs外泌体通过转运抗凋亡的miR-21-5p来减轻小鼠肺缺血/再灌注损伤。外泌体中的miR-21-5p通过靶向肺组织中的PTEN和PDCD4，减少氧化应激诱导的细胞凋亡。
最近的研究发现，超表达miR-30b-3p的MSCs来源的外泌体，可抑制血清淀粉样蛋白A3(SAA3)。
在盲肠结扎和穿孔诱导的脓毒症模型中，外泌体miR-146a增强了IL-1β预处理MSC的治疗效果，进一步发现IL-1β刺激MSCs，可以上调MSCs miR-146a的表达， 后者可以被包装在外泌体中。然后，这一外泌体miR-146a转移到巨噬细胞，导致M2极化，最终导致脓毒症小鼠存活率增加。因此，利用特异性miRNA的过表达修饰MSCs的外泌体，是发展ARDS治疗的一个有希望的新方向。
急性呼吸窘迫综合征
ARDS是由于吸烟、溺水、误吸、败血症、创伤、缺血和接触毒素等因素引起的直接或间接性肺损伤引起的急性全身性炎症反应。严重的炎症反应引起血管通透性改变，导致急性肺水肿。
ARDS病理过程主要有三个阶段:渗出期、增殖期和纤维增生期。
在渗出期，肺损伤引起的炎症级联反应，肺泡-毛细血管屏障功能障碍，导致肺泡上皮和肺毛细血管内皮细胞通透性增加，是渗出期的特征。肺组织病理表现为弥漫性肺泡损伤伴随渗出，微血管损伤伴继发肺水肿，肺泡1型(AT1)上皮细胞坏死，炎症细胞聚集，释放活性介质。肺泡炎症主要是由多形核中性粒细胞、单核细胞和巨噬细胞引起的。其他促炎机制也参与其中，如肺细胞、炎性细胞和成纤维细胞释放大量促炎细胞因子。
在增殖期，肺泡内形成透明膜，并伴有炎症细胞的浸润，包括T淋巴细胞、中性粒细胞和巨噬细胞。在炎症和氧化应激导致的严重损伤后，细胞外基质沉积于肺泡内，并伴随持续的慢性炎症。炎症级联作用在与ARDS密切相关的细胞凋亡、增殖、迁移等过程中起关键作用。持续损伤与损伤不能及时修复，是ARDS纤维增殖期的主要病理表现。
在此纤维增殖期中，有成纤维细胞增殖、AT2细胞增生和肺组织修复。受损肺泡上皮的修复机制尚不完全清楚，它包括AT2细胞的增生，AT2细胞沿基底膜迁移形成新的上皮屏障，与细胞外基质和包括肺泡巨噬细胞在内的其他细胞发生复杂的相互作用，导致某些病例的肺结构和功能发生明显改变。肺部计算机断层扫描(CT)可以在ARDS的纤维增殖期，检测到致密纤维化和蜂窝状结构。
与ARDS相关的严重性疾病会引发呼吸机获得性肺炎、急性心肌梗死和急性肺栓塞的高风险。
细胞膜外囊泡
本发明提供了一种间充质干细胞(包括脂肪间充质干祖细胞，脐带间充质干细胞和胎盘羊膜间充质干细胞)来源的细胞膜外囊泡。
在本发明中，采用本发明特定的优化工艺所制备的细胞膜外囊泡，具有特定的粒径分布，并且与PEG交联形成亲水性粒子，不仅极其适合通过气雾剂形式施用于并作用于肺部(尤其是下呼吸道)，而且能够在水溶液中长期稳定存在，且具有优异的分散性(均优于细胞本身)。
为了便于理解，申请人提供了示意性的图10。如图10所示，代表性的细胞膜外囊泡、可溶性细胞因子与PEG交联形成亲水性粒子。
在另一优选例中，用本发明所描述的分离方法和工艺获得的细胞膜外囊泡，粒径在50-150nm范围内。细胞膜外囊泡、可溶性细胞因子与PEG交联形成亲水性粒子，粒径小于3微米(如约1-3微米或0.5-3微米)，在水溶液中稳定性、分散性均优于细胞本身。利用这种细胞膜外囊泡、可溶性细胞因子与PEG交联形成亲水性粒子，分别与生理盐水、人工泪液、透明质酸等溶液配制的雾化吸入液、滴眼液、滴鼻液和外用，有利于局部粘膜细胞吸收利用这些细胞膜外囊泡和可溶性活性细胞因子。其在体内的生物分布与单纯膜外囊泡一致。
在本发明中，宜采用间充质干细胞来制备细胞膜外囊泡的细胞。代表性的间充质干细胞例子包括(但并不限于)：脂肪间充质干细胞，脐带间充质干细胞、胎盘羊膜间充质干细胞或其组合。
药物组合物和雾化吸入制剂
本发明还提供了一种药物组合物，所述的药物组合物含有(a)人的体细胞衍生的活性物质,所述的活性物质为人的体细胞所产生的细胞膜外囊泡；和(b)药学上可接受的载体。
在另一优选例中，所述的药物组合物是无细胞的药物组合物。
在本发明中，优选地，所述的活性成分还包括可溶性活性细胞因子，这些可溶性活性细胞因子主要存在于细胞膜外囊泡中。此外，一些可溶性活性细胞因子可能存在于细胞膜外囊泡之外。一些可溶性活性细胞因子还可被PEG修饰。
在本发明中，一种特别优选的药物制剂是雾化吸入制剂。
制备方法
本发明还提供了制备细胞膜外囊泡的方法。
一种典型的方法是采用间充质干细胞(包括脂肪间充质干(祖)细胞，脐带间充质干细胞和胎盘羊膜间充质干细胞)作为原料，来制备本发明的细胞膜外囊泡。
以脂肪间充质干(祖)细胞为例，一种典型的制备方法包括步骤：
(S1)培养细胞(如脂肪间充质干(祖)细胞)，使其达到预定的汇合度(如75-90％)；
(S2)在适合产生EV的条件下，继续培养所述的细胞一段时间T1；其中，所述的T1通常为24-72小时，较佳地30-60小时；
(S3)从培养体系中去除细胞，从而分离获得含细胞膜外囊泡的培养液，即为“条件培养液”(conditioned medium)”；
(S4)将所述的条件培养液”(conditioned medium)与聚乙二醇(PEG)进行混合，形成第一混合物，并放置一段时间T2，从而形成经PEG修饰的细胞膜外囊泡；其中， 所述的T2通常为6-60小时，较佳地12-48小时；
(S5)对上一步骤的第一混合物进行离心，使得所述的经PEG修饰的细胞膜外囊泡沉淀，并弃去上清，获得经PEG修饰的细胞膜外囊泡沉淀；
(S6)对上一步骤获得的经PEG修饰的细胞膜外囊泡沉淀进行重悬，从而获得第一重悬混合物；
(S7)对第一重悬混合物进行离心，使所述的经PEG修饰的细胞膜外囊泡沉淀，并弃去上清，获得经PEG修饰的细胞膜外囊泡沉淀；
(S8)对上一步骤中获得的经PEG修饰的细胞膜外囊泡沉淀进行重悬，从而获得可医用的细胞膜外囊泡制剂(即PEG-EVs-factors制剂)。
一种示意性的从步骤S4到S8的工艺路线，示于图11中。
GMP条件生产细胞膜外囊泡的工艺规模
一个HyperFlask细胞工厂可以生产500ml条件培养基的细胞膜外囊泡，预计分离5×10 10—10×10 10细胞膜外囊泡总量。
目前实验室产能一个生产操作单元可以分离两个细胞工厂共计800-1200ml条件培养基的细胞膜外囊泡，预计分离2-5×10 11细胞膜外囊泡，按照一个病人2-5×10 9的使用量，一个生产操作单元一批次生产量可以满足100-250个病人的使用量。
发明的主要优点包括：
(a)适合规模化生产，尤其是适合临床工业化规模生产。一个HyperFlask细胞工厂可以生产500-600ml条件培养基的细胞膜外囊泡，预计分离2-5×10 11细胞膜外囊泡总量。以GMP实验室产能，一个生产操作单元可以分离两个细胞工厂所产生的800-1200ml条件培养基的细胞膜外囊泡，预计可分离纯化2-5×10 11个细胞膜外囊泡。按照一个病人2-5×10 9的局部使用剂量，一个生产操作单元一批次生产量可以满足100-250个病人的使用量。
(b)稳定性好：无需DMSO等对人体有害的防冻液，在低温(-20℃)和超低温(-80℃至-196℃)条件下保存36个月或更长时间，其膜结构和内容物性质稳定。
(c)分散性高：在药学可接受的载体，特别是在等渗氯化钠水溶液中分散性好。冻融后置于4℃，24小时及以上，不会析出和沉淀，显著优于细胞类制剂。
(c)本发明的药物组合物在水溶液中均匀分散的、小于3微米的亲水粒子，经雾化吸入给药，可以异乎寻常地高效进入了呼吸道，尤其是下呼吸道。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor  Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
材料
脂肪间充质干(祖)细胞：对于经过伦理审查备案的、签署知情同意书的、经过严格实验室检查符合脂肪组干细胞产品建库入组标准的男性或女性健康志愿者，经过腹壁穿刺抽脂、或腹壁手术切取脂肪，置于细胞保存液中保存的、低温运输至合格的GMP实验室，经过分离、纯化、增殖处理，所获得的脂肪间充质祖细胞，即工作库细胞(中间产品)。
通用方法
1.培养基预处理
将含有5％EliteGro的αMEM，于4℃条件下，120,000g离心6h。
2.PEG沉淀法获得细胞膜外囊泡
用PBS配制12％PEG6000，用0.22μm的过滤器过滤后，按照1:1体积比加入到处理后的条件培养基，置于4℃过夜孵育后，4℃，3,000g离心1h，去掉上清。加入预冷的PBS重悬沉淀，4℃，12,0000g超速离心70min，吸去上清，加入适量等渗氯化钠溶液重悬沉淀即为细胞膜外囊泡。
3.粒子直径分布和粒子浓度的测定
取适量样品，稀释300倍，用ZetaView测定样品的粒子直径分布和粒子浓度。
4.蛋白浓度的测定
取适量样品，以BCA法测定蛋白浓度，加入5×SDS上样缓冲液，100℃加热5min，将同蛋白量样品上样于SDS聚丙烯酰胺凝胶，100V电泳至染料迁移至凝胶底部，250mA，90min转移至PVDF膜上。用TBST缓冲液配制5％脱脂牛奶作为封闭液和一抗二抗稀释液，封闭PVDF，用一抗孵育，分别为阳性蛋白CD63、CD81、TSG101，阴性蛋白CANX，并用相应二抗孵育后，加入显影液观察蛋白条带情况。
实施例1
含细胞膜外囊泡的培养液的制备(条件培养基的制备)
取2×10 7个脂肪间充质干细胞(P4代)，接种于含5％EliteGro的αMEM基础培养基中，在HyperFlask培养瓶中培养，培养条件为37℃，5％CO 2。
当培养至细胞融合度达到约80％，换液，使用预处理的培养基进行培养，条件为 37℃，5％CO 2，培养48h。
收集培养上清，3,000g离心15分钟后取上清，10000g，4℃条件离心30分钟。取上清，即为含细胞膜外囊泡的培养液，以下称为“条件培养基”(conditioned medium)。
实施例2
细胞膜外囊泡的制备
在本实施例中，分别通过PEG沉淀法和超速离心法来制备细胞膜外囊泡。
超速离心法：
取条件培养基(实施例1)，于4℃，120,000g超速离心70min后，用预冷的PBS重悬，再次120,000g超速离心70min后，用适量等渗氯化钠溶液重悬沉淀，获得细胞膜外囊泡。
PEG沉淀法：
用PBS配制浓度为8％、12％、16％、或20％PEG6000，用0.22μm的过滤器过滤后，按照1:1体积比加入到处理后的条件培养基(实施例1)，置于4℃过夜孵育后。
4℃，3,000g离心1小时，弃去上清。加入预冷的PBS重悬沉淀，4℃，120,000g超速离心70分钟，弃去上清，加入适量等渗氯化钠溶液重悬沉淀，获得细胞膜外囊泡。
性能测定：
将分别添加含8％PEG 6000、12％PEG 6000、16％PEG 6000、20％PEG 6000的分离出的细胞膜外囊泡按照相同的总蛋白量上样，进行CD63鉴定。
结果如图1A所示，12％PEG 6000和16％PEG 6000分离出的细胞膜外囊泡的CD63表达量最高。因此，在后续实验，使用12％PEG 6000。
此外，将使用12％PEG 6000分离200ml培养基所获得的细胞膜外囊泡和超速离心分离350ml培养基所获得的细胞膜外囊泡，按照同体积和同蛋白量上样进行CANX、TSG101和CD81鉴定和NTA检测。
结果如图1B至图1D所示。结果表明，无论是同体积还是同蛋白做Western鉴定，PEG 6000分离出的细胞膜外囊泡的目的蛋白量都远大于超速离心法分离出的细胞膜外囊泡的量。
NTA结果显示，PEG 6000分离出的细胞膜外囊泡的浓度为1.35×10 8细胞膜外囊泡/ml培养基，超速离心分离出的细胞膜外囊泡的浓度为6.8×10 7细胞膜外囊泡/ml培养基。
PEG 6000分离出的细胞膜外囊泡粒径大小为126.6±2.09nm，超速离心分离出 的细胞膜外囊泡的粒径大小为137.8±3.8nm。
这些实验结果证明，12％PEG 6000分离出的细胞膜外囊泡产量比超速离心分离出的产量高的多，且PEG并不影响细胞膜外囊泡的粒径大小。
实施例3
成纤维细胞摄取细胞膜外囊泡
使用PKH-67，将PEG 6000分离出的细胞膜外囊泡进行标记。
图2可以发现，PEG 6000分离出的细胞膜外囊泡并不影响成纤维细胞对细胞膜外囊泡进行摄取。
实施例4
细胞膜外囊泡的稳定性和性能测定
将分离制备完成的细胞膜外囊泡，用生理盐水配制成水溶液，分别置于4℃、-20℃和-80℃，贮存9周。9周以后，将置于3个温度的细胞膜外囊泡做NTA检测和相关性能测定。
NTA检测结果如图3和表1所示。
表1 NTA测量的粒子数
Figure PCTCN2021080869-appb-000001
结果表明，3个不同温度贮存9周的细胞膜外囊泡粒径数量与第0天初次分离的细胞膜外囊泡数量没有显著性减少(图3)。
Western蛋白电泳的结果显示，在-20℃和-80℃条件下，储存9周的细胞膜外囊泡的特异性标志物CD81和TSG101都表达稳定。4℃条件下，储存3周的细胞膜外囊泡的特异性标志物TSG101能够稳定表达，从第5周开始，该标志物的表达量开始下降(图4)。
在透射电镜下，于-80℃贮存1周的细胞膜外囊泡，呈“茶托样”(图5A)。在常温复融半小时以后，该细胞膜外囊泡在透射电镜下，仍然呈“茶托样”(图5B)，没有显著性变化。
在-80℃贮存1周的细胞膜外囊泡和常温复融半小时后的细胞膜外囊泡经NTA检测，发现其细胞膜外囊泡的粒径浓度(图6A)和大小(图6B)没有显著性差异。
实施例5
含细胞膜外囊泡和细胞因子的冻干制剂的多种细胞因子检测
在本实施例中，对本发明方法制备的膜外囊泡进行细胞因子检测。测试样品包括：
样品1:基础培养基的冻干粉(阴性对照)，4℃保存4周；
样品2:干细胞培养液冻干粉(阳性对照)，4℃保存4周；
样品3:细胞膜外囊泡冻干粉，常温保存4周；
样品4:细胞膜外囊泡冻干粉，4℃保存4周；
样品5:细胞膜外囊泡冻干粉，-20℃保存4周；
针对多个活性细胞因子的检测结果如表2所示。
表2细胞因子的检测结果
Figure PCTCN2021080869-appb-000002
结果表明，本发明所描述的分离纯化制备工艺方法所制备的细胞膜外囊泡含有丰富的活性细胞因子。在-20℃和-4℃条件下保存4周，细胞因子的稳定性很好。
实施例6
人源脂肪间充质祖细胞膜外囊泡抑制LPS诱导巨噬细胞活化
为了证实人源脂肪间充质祖细胞膜外囊泡对脂多糖(LPS)诱导小鼠Raw264.7巨噬细胞炎症，具有抗炎作用，在本实施例中，首先以LPS诱导该巨噬细胞发生炎症为模型，体外研究人源脂肪间充质祖细胞膜外囊泡对其活化水平的影响。具体方法如下：
首先设置了四组试验，分别是不做任何药物处理的对照组(control)，0.1μg/ml LPS单独处理组，0.1μg/ml LPS和5μg/ml地塞米松(Dex)共同处理组，以及0.1μg/ml LPS和适当浓度的人源脂肪间充质祖细胞膜外囊泡(haMPCs-Exo)处理组。
向12孔培养板的每个孔中加入1ml的Raw264.7巨噬细胞悬液(1×10 4细胞/孔)，置于CO 2恒温培养箱中培养(5％CO 2,37℃)1h，然后按照上述试验分组进行药物处理，并在CO 2恒温培养箱中培养(5％CO 2,37℃)中继续培养4小时。最后，取出培养板，在倒置显微镜下观察活化的小鼠Raw264.7巨噬细胞的形态变化情况，并在20倍物镜下随机选取视野进行拍照记录和统计分析。
结果如图7所示，与对照组(control)组相比，单独使用LPS诱导4小时后，活 化的巨噬细胞(促炎症型巨噬细胞)的形态呈分枝状(图7A)，且统计分析发现其活化数目也显著增加(图7B)，这表明已成功建立了体外的LPS诱导巨噬细胞炎症模型。
与LPS单独处理组相比，经LPS和地塞米松处理4小时后，呈分枝状的巨噬细胞明显减少(图7A)，且统计分析发现其活化的巨噬细胞数被显著抑制(图7B)。
同样地，经LPS和人源脂肪间充质祖细胞膜外囊泡处理4小时后，呈分枝状的巨噬细胞也明显减少(图7A)，且统计分析发现其活化的巨噬细胞数也被显著抑制(图7B)。
因此，这些结果表明，人源脂肪间充质祖细胞膜外囊泡能够抑制LPS诱导的巨噬细胞活化，进而削弱了促炎症型巨噬细胞的形成。
实施例7
人源脂肪间充质祖细胞膜外囊泡抑制LPS诱导巨噬细胞促炎症因子基因表达水平
在本实施例中，为了证实人源脂肪间充质祖细胞膜外囊泡对LPS诱导小鼠Raw264.7巨噬细胞的炎症反应具有抑制作用，采用实时荧光定量PCR检测其主要炎症因子基因的表达水平。其具体操作方法基本同实施例1所述，但药物处理时间为24小时。总RNA提取方法采用碧云天RNA提取试剂盒说明书进行，而实时荧光定量PCR检测则采用TaqMan探针法进行靶基因和内参基因的同时检测，并按2 -ΔΔCT方法进行相对定量分析。
结果如图8所示，与对照组相比，LPS单独处理24小时后，小鼠Raw264.7巨噬细胞的促炎症因子基因表达水平显著增加，然而在LPS和地塞米松共同处理24小时后，TNF-α、IL-1β和IL-6的基因表达水平均极显著降低(图8A、B和C)。
同样地，经LPS和人源脂肪间充质祖细胞膜外囊泡处理24小时后，TNF-α、IL-1β和IL-6的基因表达水平也都极显著降低，基本达到了地塞米松处理后的水平(图8A、B和C)。
因此，这些结果表明，人源脂肪间充质祖细胞膜外囊泡能够显著抑制LPS诱导巨噬细胞促炎症因子基因的表达水平，进而抑制其炎症反应。
实施例8
人源脂肪间充质祖细胞膜外囊泡抑制LPS诱导巨噬细胞促炎症因子释放
在本实施例中，为了进一步确认人源脂肪间充质祖细胞膜外囊泡能够抑制LPS诱导小鼠Raw264.7巨噬细胞炎症反应，采用ELISA法检测其分泌出的炎症因子水平。其具体操作方法同实施例2所述，但药物处理时间为24小时。实验方案参见图9A。ELISA法检测巨噬细胞炎症因子的操作按照碧云天TNF-α和IL-6试剂盒说明书进行，然后进行统计分析。
结果表明，与对照组相比，LPS单独处理24小时后，小鼠Raw264.7巨噬细胞释放的促炎症因子TNF-α和IL-6均显著增加(图9B)，然而在LPS和地塞米松共同处理24小时后，TNF-α和IL-6水平均极显著降低(图9B)。
同样地，经LPS和人源脂肪间充质祖细胞膜外囊泡处理24小时后，TNF-α和IL-6水平也都极显著降低，达到了地塞米松处理后的水平(图9B)。其中，膜外囊泡对于IL-6的抑制作用更为优异，提示其在抑制新冠病毒肺炎细胞因子风暴干预治疗中有更好的应用前景。
因此，上述数据表明，人源脂肪间充质祖细胞膜外囊泡能够显著抑制LPS诱导巨噬细胞促炎症因子的释放，进而抑制其促炎症反应。
实施例9
雾化吸入荧光标记的EV在裸鼠体内生物分布研究方案
在本实施例中，研究人源脂肪间充质祖细胞外泌体经雾化吸入后，在小鼠肺组织中的分布情况及其持续时间。采用荧光标记外泌体示踪法，对其进行探究。
使用PKH67标记的人源脂肪间充质祖细胞外泌体(1×10 6细胞膜外囊泡/g)，经雾化处理30分钟并吸入裸鼠(N＝3)肺部后，分别选取雾化吸入后的2h、4h、6h、8h、12h和24h时间点，采用活体荧光成像技术对PKH67标记的外泌体在裸鼠肺组织中的分布情况进行观察，并分析其在裸鼠肺部的滞留时间。
实施例10
雾化吸入EV治疗感染性肺损伤动物模型有效性研究方案
在本实施例中，研究人源脂肪间充质祖细胞外泌体经雾化吸入后，治疗耐多药铜绿菌(PA)诱导的小鼠肺炎模型的有效性。分别设置PBS诱导小鼠的空白对照组，PA诱导小鼠肺炎组，人源脂肪间充质祖细胞外泌体雾化吸入治疗PA诱导小鼠肺炎组，以及小鼠成纤维细胞外泌体治疗小鼠肺炎组。每组均为6只8-10周龄的C57BL/6野生型雄性小鼠，且治疗组中的每只小鼠外泌体用量均按1×10 6细胞膜外囊泡/g计算。耐多药铜绿菌诱导4小时后，进行外泌体雾化吸入，每日雾化1次，持续5天。之后，收集血液样本用于外周血白细胞和中性粒细胞计数及细菌负荷测量。收集肺泡灌洗液，用于中性粒细胞计数、细菌负荷测量，以及相关促炎症因子水平测定。另外，测定肺组织细菌负荷，对肺组织进行HE染色的形态学观察。
实施例11
雾化吸入EV治疗新冠病毒肺炎急性肺损伤临床试验方案
在本实施例中，初步探索异体脂肪间充质干细胞外泌体经雾化吸入治疗新型冠状病毒肺炎(Novel Coronavirus Pneumonia，NCP)重型和危重型患者的安全性和有效性。
拟定纳入30例重型和危重型NCP受试者，分为低、中、高三个剂量组爬坡。所有受试者除ICU常规治疗外，联合给予雾化吸入异体脂肪MSCs-Exo治疗，每次2-8×10 8细胞膜外囊泡/3ml，每日1次，连续5天。记录并分析28天内的不良反应发生率和临床改善时间，以及患者脱机例数，ICU护理天数，幸存者机械通气持续时间和死亡率等。此外，通过每日SOFA评分，相关生化指标的检测和肺部影像学观察，以及患者呼吸道标本中新冠病毒转阴时间，对患者经治疗后的症状改善进行评判。本研究入组标准主要包括1)年龄18-75岁，男女不限，且本人或家属自愿加入并签署知情同意书；2)RT-PCR检测呈阳性或明确诊断为新型冠状病毒肺炎患者；3)符合重型和危重型患者的诊断标准。排除标准主要包括1)相关病毒携带者或较严重过敏体质患者，其他病毒引起的肺炎患者；2)肺癌或长期使用免疫抑制药物的患者；3)进行血液透析或腹膜透析的患者，以及肝功能异常患者；4)正在使用ECMO或高频振荡通气的患者；5)孕期、哺乳期或半年内计划孕育的患者；6)研究者评判不能参与研究的或未能理解和执行该方案的患者。
讨论
对于急性呼吸窘迫综合征的治疗，一些可供选择的细胞疗法包括胚胎干细胞(embryonic stem cells,ESCs)治疗、体外诱导的多能干细胞(induced pluripotent stem cells,iPSCs)治疗、间充质干细胞(Mesenchymal stromal/stem cells，MSCs)治疗、肺上皮祖细胞(Pulmonary epithelial progenitor cells，EpPCs)治疗和内皮祖细胞(Endothelial progenitor cells，EnPCs)治疗等。
MSCs是研究最多的一种干细胞疗法。MSCs是一种可自我更新和增殖的多能性干细胞，能够在体外抑制免疫反应，并具有分化成肺泡2型细胞(AT2细胞)的潜能。MSCs介导的炎症抑制和MSC相关的肺修复和再生作用，是其有可能成为治疗ARDS、肺炎、哮喘、慢性阻塞性肺疾病(COPD)和肺间质纤维化(IPF)等肺脏疾患的主要原因。
如前所述，ARDS是一种严重的临床综合征，是肺泡上皮屏障破坏所引起的，伴有间质性水肿和炎性细胞浸润，导致进行性急性呼吸衰竭。尽管合成的皮质类固醇激素、表面活性剂、吸入型一氧化氮、抗氧化剂、蛋白酶抑制剂，以及各种其他抗炎治疗，如辛伐他汀和布洛芬等许多药物已被用于治疗ARDS，但没有一种药物能显著降低ARDS的死亡率，ARDS的死亡率仍然维持在34％-44％。
最近几项临床前研究表明，MSC及其旁分泌细胞因子和细胞外微囊泡可被认为是治疗ARDS的一种新的、有效的治疗手段。ARDS通常是严重脓毒症的并发症，尤其是在革兰氏阴性菌感染后。MSCs治疗可以阻止大肠杆菌源性脂多糖(LPS)诱导的脓毒症动物模型中ARDS的发生[Curley G F,et al.Critical Care Med.2017,45(2):e202–e212.][Lee JW,et al.Stem Cell 2011,29(6):913–919.]。
在脂多糖诱导的ARDS小鼠模型，经静脉注射间MSCs能显著改善肺泡损伤和炎症 反应。MSCs通过旁分泌和白细胞介素-10(IL-10)依赖的方式，减少中性粒细胞在肺内浸润，减少肺内浸润的免疫细胞产生炎症因子TNF-α[Gupta N,et al.Journal of Immunology 2007,179,(3):1855–1863.][Mei SHJ,et al.PLoS Medicine,2007,4(9):e269.]。
此外，通过角化细胞生长因子(KGF)、血管内皮生长因子(VEGF)和肝细胞生长因子(HGF)的产生，MSCs促进了AT 2型细胞的再生，阻止内皮细胞的凋亡，促进ARDS损伤的肺上皮屏障的修复[Lee JW,et al.Proceedings of the National Academy of Sciences of the United States of America2009,106(38):16357–16362.][Hu S,et al.Stem Cell Research&Therapy2016,7(1):66.]。经MSCs处理的动物模型，炎症损伤减少，水肿减轻，氧合改善，生存期延长。通过增加巨噬细胞产生抗炎因子IL-10，MSCs以依赖PGE-2的方式来保护自身免受脓毒症相关ARDS的侵袭。
经静脉输注骨髓MSCs治疗ARDS的早期临床试验已经完成。[J.G.Wilson JG,et al.Lancet Respir.Med.2015,3(1):24–32.][Zheng G,et al.Respir.Res.2014,15(1):39.]。Wilson等临床研究(NCT01775774)报告，在9例中重度ARDS患者中，单次静脉输注同种异体骨髓MSCs(100万、500万、1000万cells/kg)，具有良好的耐受性。9例接受异体骨髓MSCs治疗的患者均未观察到不良反应和剂量限制性毒性。在这项I期临床试验结束后，一个随机多中心的II期临床试验从2014年到2018年在美国进行(NCT02097641)。该试验招募60名成年ARDS患者，分别给予单剂量同种异体骨髓MSCs(1000万cells/kg)或安慰剂治疗。结果尚未见报告。
在另一项静脉注射同种异体脂肪组织来源的MSCs治疗ARDS的I期临床试验(NCT01902082)，纳入12例成人ARDS患者，一次静脉注射同种异体脂肪MSCs(100万cells/kg)，但AT-MSCs治疗并不能显著降低ARDS患者血清炎性细胞因子(IL-6和IL-8)水平，也不能改善ARDS患者的肺功能，预示这是一个安全但无效的治疗研究。
最近研究发现，MSCs产生的微囊泡(extracellular vesicles,EVs)，促进肺泡巨噬细胞的吞噬活性，从而减轻革兰氏阴性大肠杆菌诱导的细菌性肺炎[Monsel A,et al.Am J Respir Crit Care Med.2015,192(3):324–336.]。此外，MSCs产生抗菌蛋白，能够直接抑制炎症肺内的细菌生长。
气管内注射MSCs可显著减轻肺损伤和炎症反应，并通过以脂钙素-2依赖的方式促进细菌清除，从而提高实验动物细菌性肺炎的存活率。LPS诱导的MSCs中TLR-4的激活，增强了脂钙素-2的分泌，而脂钙素-2可以结合细菌的铁载体，减少铁的吸收，抑制细菌的生长。与这些发现相一致的是最近报道的研究结果：在细菌性肺炎的实验模型中，Gupta等[Gupta N,et al.Thorax2012，67(6)：533–539]发现MSCs中TLR-4的突变显著地削弱了它们的治疗效果。因此，预示一种潜在的新的基于细胞的治疗方法，可能消除肺部的抗生素耐药性革兰氏阴性菌株。
本发明的研究表明，间充质干细胞膜外囊泡的显著优势。干细胞在多种疾病的治 疗中具有巨大的潜力。干细胞治疗效果很大程度上依赖于其旁分泌的细胞因子和膜外囊泡(extracellular vesicles,EVs)或外泌体(exosomes)所介导的。
EVs是一种纳米大小的膜结构囊泡，具有细胞与细胞间通讯的中介功能。MSCs来源的EVs包含细胞因子、生长因子、信号脂质、mRNA、miRNA/siRNA等物质。
越来越多的研究证据表明，MSC-EVs可能代表一种新的无细胞治疗方法，与现有的MSCs疗法相比，具有明显的优势：1)在体内无肿瘤形成风险；2)较低的免疫原性；3)EVs可以来源于内源性MSCs，也可以由异体人MSCs培养过程中的上清液(条件培养基)经分离纯化获得，可以满足临床级规模化生产和纯化，批次产量高，便于批次质量控制检测；4)可以对产生EVs的特定增殖代次的种子干细胞进行体外修饰，富集更加特异性的EVs，用于不同的治疗目的；5)单个悬浮状态的MSC直径(18-40μm)是红细胞直径(6μm)的3-7倍，易于沉积和粘连形成微凝块。经静脉注射后，有堵塞微血管的风险。EVs为50-500nm直径范围内的纳米膜结构囊泡，在水溶液中分散性好，易于制成注射制剂；6)EVs具有很好的膜结构稳定性,对低温冻存和冻融过程有较强的耐受性；7)因EVs的膜结构特点和纳米粒子特性，可被靶组织细胞主动吞噬摄取，进而获得EVs携带的生物信息和(或)活性药物，以发挥EVs对细胞的直接信号传导和药物靶向递送功能。8)EVs可以被装载某些活性药物，成为一种天然的药物递送系统。9)多个研究小组已经报道了MSC衍生的EVs的治疗潜力。MSCs-EVs有可能作为未来ARDS的生物标志物和治疗制剂。MSCs-EVs在ARDS恢复过程中似乎也起着关键作用。
多项研究发现，MSC来源的微囊泡(microvesicles,MVs)经气道或静脉给药途径具有治疗优势。Zhu等的研究表明，在大肠杆菌内毒素诱导的肺损伤中，气管内灌注MSCs衍生的MVs可以减少肺组织细胞外含水量，减少肺水肿，降低肺泡膜对蛋白质通透性。MSC-MVs还减少了中性粒细胞的流入，降低了巨噬细胞炎症蛋白-2在肺泡灌洗液(BAL)中的水平。
MSCs通过EVs介导的线粒体转移来调节ALI中的巨噬细胞。MSC-MVs中含有大量的血管生成素-1，而巨噬细胞上MSCs的免疫调节特性部分是通过向巨噬细胞转移血管生成素-1mRNA介导的。MSCs外泌体(exosome)通过肝细胞生长因子(HGF)，减少内皮细胞凋亡、增加IL-10的产生和减少IL-6的产生，部分地改善了LPS诱导的小鼠急性肺损伤模型的肺微血管通透性。
MSCs-MVs可恢复肺泡膜紧密联接的完整性，也可减低IL-1β、TNF-α和IFN-γ对人肺微血管内皮细胞渗透性增强作用。抗CD44和血管生成素-1siRNA预处理，可以消除MSCs-MVs的这种治疗作用，表明CD-44和血管生成素-1mRNA转移，参与了MSCs-MVs的修复治疗机制。可以通过预处理MSCs来丰富MVs亚群的表达，从而增加其治疗潜力。
基于本发明人的研究，提供了以下各项发明：
发明项1:一种含有人体细胞衍生的活性物质的药物制剂，该制剂的成分的稳定性好，分散度高，易于被人体除皮肤以外的局部所吸收，适用于预防和治疗各种原因导致的炎症和损伤。
发明项2:发明项1所述的人体细胞衍生的活性物质，其中，包括但不限于人体组织、骨髓和血液来源的干细胞、祖细胞和免疫细胞所产生的细胞膜外囊泡与可溶性活性细胞因子。
发明项3:发明项1所述的药物制剂，其中，包括但不限于雾化吸入液、滴眼液、滴鼻液。
发明项4:发明项1所述的药物制剂稳定性好，其中，包括但不限于在常温条件下保存、-20℃以下保存、-70℃以下低温条件下保存、-135℃以下低温条件下保存、-196℃低温条件下保存，其活性物质的稳定性显著优于相同条件下含有活细胞制剂的保存时间。
发明项5:发明项1所述的药物制剂分散度高，其中，在0-25℃条件下，静置6-24小时，呈现无色透明，且无肉眼可见的絮状物和沉淀物。
发明项6:发明项1所述的药物制剂易于在人体除皮肤以外的其他部位使用，其中，这些部位包括但不限于眼球结膜、眼睑结膜、视网膜、口腔、鼻腔、上呼吸道、下呼吸道、胃肠道、血管内皮细胞、肺泡1型上皮细胞、肺泡2型上皮细胞、单核巨噬细胞、中性粒细胞、树突状细胞、抗原递呈细胞、T淋巴细胞、纤维母细胞、中枢神经细胞、周围神经细胞及其末梢神经纤维。
发明项7:发明项1所述的药物制剂，适用于预防和治疗各种原因所致的炎症和损伤，其中，包括但不限于病毒感染性炎症、细菌感染性炎症、真菌感染性炎症、自身免疫反应性炎症、缺血性损伤、化学性损伤、物理性损伤。
发明项8:发明项2所述的人体组织、骨髓和血液来源的干细胞、祖细胞和免疫细胞，其中，包括但不限于人体脂肪组织来源的间充质干细胞、人体肺组织来源的肺泡上皮祖细胞、人体脐带华通氏胶来源的间充质干细胞、人体子宫内膜组织来源的间充质干细胞、人体胎盘组织来源的干细胞、人体胎盘羊膜来源的上皮细胞、人体血液中的单个核细胞、人体骨髓组织中的造血干细胞、人体骨髓组织中的间充质干细胞、人体骨膜来源的间充质干细胞、人体皮肤组织来源的角质细胞、人体血液组织来源的树突状细胞、人体血液组织来源的CD4阳性T淋巴细胞、人体血液组织来源的血小板。
发明项9:发明项2所述的人体细胞衍生的细胞膜外囊泡，其中，包括但不限于由脂质双分子层膜结构包绕形成的纳米囊泡，直径在30-1000纳米之间、内含丰富的不同种类的、具有特定功能的微小核糖核酸(miRNA)、小核糖核酸(sRNA)、非编码的DNA片段、转运核糖核酸t-RNA、可溶性细胞因子、生长因子、其他蛋白质。
发明项10:发明项2所述的人体细胞衍生的可溶性活性细胞因子，其中，包括但不限于由可溶性的游离的脂蛋白质分子、糖蛋白分子、生物信号分子、组织生长因子 B(TGFβ)、干细胞生长因子(HGF)血管内皮细胞生长因子(VEGF)、表皮细胞生长因子(KGF)、白细胞介素-10(IL-10)、白细胞介素-1受体拮抗剂(IL-1βA)。
发明项11:发明项2和发明项10中所述的人体细胞衍生的可溶性活性细胞因子，其中，包括但不限于由自然状态下培养的人体细胞旁分泌所产生的、用特定基因修饰人体细胞超表达产生的。
发明项12:发明项2和发明项9所述的细胞膜外囊泡，其中，特异性表达膜蛋白标志物CD9、CD63、CD81和TSG101，不表达CANX。细胞膜外囊泡其直径优选为50-500nm。
发明项13：发明项2和发明项8所述的人体组织、骨髓和血液来源的干细胞、祖细胞和免疫细胞，其中，这些细胞可以从人体组织直接分离纯化所获得，或这些细胞可以从人体组织直接分离纯化后，再经过GMP实验室最小程度的操作，使这些细胞增殖后所得，或这些细胞在GMP实验室经过特定基因修饰、特定基因编辑、特定基因转导、特定微小核糖核酸miRNA的导入所得，或这些细胞在GMP实验室经过特殊培养条件的预处理所得。
发明项14：发明项2和发明项8所述的人体组织、骨髓和血液来源的干细胞、祖细胞和免疫细胞，其中，这些细胞优选为脂肪来源的间充质干细胞、胎盘羊膜来源的间充质干细胞。
发明项15：发明项2、发明项8、发明项13、发明项14中所述的人体组织、骨髓和血液来源的干细胞、祖细胞和免疫细胞，进一步优选为脂肪来源的间充质干(祖)细胞、胎盘羊膜来源的间充质干细胞。
发明项16：发明项15所述的进一步优选为脂肪来源的干(祖)细胞，其中，经过伦理审查备案的、签署知情同意书的、经过严格实验室检查符合脂肪组干细胞产品建库入组标准的男性或女性健康志愿者，经过腹壁穿刺抽脂、或腹壁手术切取脂肪，置于专利保护的细胞保存液中保存的、低温运输至合格的GMP实验室，经过专利保护的分离、纯化、增殖的脂肪间充质祖细胞产品的工作库细胞(中间产品)。
发明项17：发明项15和发明项16所述的进一步优选的人体脂肪组织来源的间充质祖细胞，其中，这种特定的人体脂肪组织来源的间充质干(祖)细胞是在GMP生产实验室，按照中间产品生产工艺流程生产的、符合各种病原学检测阴性标准的、符合特定表面标志物如CD73阳性、CD90阳性、CD105阳性细胞百分率大约98％，CD34阳性、CD45阳性和HLA-DR阳性的细胞百分率小于2％的、符合抗生素残余和血清及血清替代物残余检测标准的、P1-P6代脂肪间充质干(祖)细胞，进一步优选为P3-P4代脂肪间充质干(祖)细胞。
发明项18：发明项15-17所述的进一步优选的脂肪组织来源的间充质干(祖)细胞为-196℃至-80℃深低温条件下储存0-36个月的，进一步优选为-196℃至-135℃，储存0-24个月的。
发明项19：发明项2和发明项9-12所述的人体细胞衍生的细胞膜外囊泡及可溶 性活性细胞因子，其中，其生产工艺是：
(1)通过接种这些类型的人体细胞于含5％EliteGro的αMEM基础培养基中，在HyperFlask培养瓶中培养，培养条件为37℃，5％CO 2,至细胞融合度达到80％，再使用特定的预处理培养基进行培养，培养条件为37℃，5％CO 2，培养48小时。
(2)收集细胞培养上清液，进行差速离心联合聚乙二醇(PEG)沉淀法分离。
发明项20：发明项19所述的生产工艺(2)中所述的差速离心联合聚乙二醇PEG沉淀，其中，使用PEG3000-PEG9000，用PBS配置为8％-30％的PEG，用0.22μm的过滤器过滤后，按照1:1体积比加入到处理后的条件培养基中，置于4℃过夜孵育后，4℃，3,000-5000g离心45-60分钟，弃去上清。加入预冷的PBS重悬沉淀，4℃，100000-120000g超高速离心60-120分钟，弃去上清，加入适量等渗氯化钠溶液，重悬沉淀，即可获得人体细胞衍生的细胞膜外囊泡及可溶性活性细胞因子。
发明项21：发明项19和发明项20所述的生产工艺获得的细胞膜外囊泡，其特征为生物标志物CD9阳性、CD63阳性、CD81阳性、TSG101阳性和CANX阴性。
发明项22：发明项19-21所述的生产工艺，其中，在GMP实验室条件下，可以放大到细胞制品临床级生产规模，其突出优势为：1个生产操作单元可以分离2个细胞工厂所获得的800-1200ml条件培养基，可分离获得2-5×10 11细胞膜外囊泡。按照一个病人2-5×10 9的细胞膜外囊泡局部使用的总剂量计算，一个生产操作单元，一个批次的生产量可以满足100-250个病人的局部使用量。100个生产操作单元，一个批次生产量可达到10000-25000人使用总量，用时5-6天。
发明项23：发明项19-22所述的生产工艺，其中，分离获得的细胞膜外囊泡、可溶性活性细胞因子与特定分子量的PEG交联成小于3微米的亲水性粒子，在-196℃至-20℃冻存条件下的稳定性和在常温水溶液中的分散性都优于现有技术。
发明项24：发明项23所述的细胞膜外囊泡、可溶性活性细胞因子及聚乙二醇PEG交联的亲水粒子，其中，包括但不限于其在等渗氯化钠溶液、低分子透明质酸溶液、人工泪液、凝胶溶液中，制备成雾化吸入液、滴眼液、滴鼻液外用凝胶制剂。局部粘膜细胞对本发明工艺所获得的细胞膜外囊泡的摄取不受PEG的影响。
发明项25：发明项1和发明项23-24所述的含有人体细胞膜外囊泡及可溶性活性细胞因子的药物制剂，优先选择制备雾化吸入剂，适于治疗感染性肺损伤。
发明项26：发明项1和发明项25所述药物制剂，其中，优选脂肪组织来源的干(祖)细胞和胎盘羊膜来源的间充质干细胞衍生的膜外囊泡和可溶性细胞因子与等渗氯化钠水溶液制备的雾化吸入液，适用于病毒，包括但不限于流感病毒、SARS冠状病毒、SARS冠状病毒2、MERS冠状病毒感染，所引起的急性肺损伤雾化吸入治疗，以达到阻止急性肺损伤炎症因子的释放、减轻肺泡内高蛋白液的渗入和肺泡上皮细胞损伤、显著提高急性肺损伤患者的抢救成功率、以及改善存活患者肺功能和生活质量。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (14)

  1. 一种药物组合物，其特征在于，所述的药物组合物含有(a)人的体细胞衍生的活性物质,所述的活性物质为人的体细胞所产生的细胞膜外囊泡；和(b)药学上可接受的载体。
  2. 如权利要求1所述的药物组合物，其特征在于，所述的体细胞选自下组：人体组织、骨髓和/或血液来源的干细胞、祖细胞、免疫细胞、或其组合。
  3. 如权利要求1-2中任一项所述的药物组合物，其特征在于，所述的药物组合物的剂型选自下组：雾化吸入剂、滴眼液、滴鼻液。
  4. 如权利要求1-3中任一项所述的药物组合物，其特征在于，所述的药物组合物具有以下特性：
    (P1)优于活细胞制剂的保存稳定性；优选地，所述的保存包括在常温和/或低温下进行保存；所述的“优于活细胞制剂的保存稳定性”指所述的药物组合物的稳定性优于相同条件下含活细胞制剂的稳定性；和
    (P2)高分散性；较佳地，当所述药物组合物为含水的液态制剂时(如以生理盐水配制的溶液时)，在0-25℃放置6-24小时，呈现无色透明，且无肉眼可见的絮状物和沉淀物。
  5. 如权利要求1-4中任一项所述的药物组合物，其特征在于，所述的药物组合物被施用于选自下组的部位：眼球结膜、眼睑结膜、视网膜、口腔、鼻腔、上呼吸道、下呼吸道、胃肠道、肺部、或其组合。
  6. 如权利要求1-5中任一项所述的药物组合物，其特征在于，所述的细胞膜外囊泡的直径优选为50-500nm。
  7. 一种制备细胞膜外囊泡的方法，其特征在于，包括步骤：
    (S1)培养人的体细胞，使其达到预定的汇合度(如75-90％)；
    (S2)在适合产生EV的条件下，继续培养所述的细胞一段时间T1；
    (S3)从培养体系中去除细胞，从而分离获得含细胞膜外囊泡的培养液，即为“条件培养液(conditioned medium)”；
    (S4)将所述的条件培养液与聚乙二醇(PEG)进行混合，形成第一混合物，并放置一段时间T2，从而形成经PEG修饰的细胞膜外囊泡；其中，所述的T2通常为6-60小时，较佳地12-48小时；
    (S5)对上一步骤的第一混合物进行离心，使得所述的经PEG修饰的细胞膜外 囊泡沉淀，并弃去上清，获得经PEG修饰的细胞膜外囊泡沉淀；
    (S6)对上一步骤获得的经PEG修饰的细胞膜外囊泡沉淀进行重悬，从而获得第一重悬混合物；
    (S7)对第一重悬混合物进行离心，使所述的经PEG修饰的细胞膜外囊泡沉淀，并弃去上清，获得经PEG修饰的细胞膜外囊泡沉淀；
    (S8)对上一步骤中获得的经PEG修饰的细胞膜外囊泡沉淀进行重悬，从而获得可医用的细胞膜外囊泡制剂。
  8. 一种细胞膜外囊泡制剂，其特征在于，所述的细胞膜外囊泡制剂是用权利要求7所述的方法制备的。
  9. 一种权利要求1-6中任一项所述的药物组合物或权利要求8所述的细胞膜外囊泡制剂的用途，其特征在于，被用于制备预防和/或治疗炎症、或损伤的药物。
  10. 如权利要求9所述的用途，其特征在于，所述的炎症选自下组：病毒感染性炎症、细菌感染性炎症、真菌感染性炎症、自身免疫反应性炎症、或其组合；
    所述的损伤选自下组：缺血性损伤、缺氧性损伤、化学性损伤、物理性损伤、或其组合。
  11. 如权利要求9所述的用途，其特征在于，所述的药物用于治疗急性呼吸窘迫综合征。
  12. 如权利要求9所述的用途，其特征在于，所述的药物为雾化吸入液。
  13. 如权利要求9所述的用途，其特征在于，所述的药物用于治疗病毒性急性肺损伤。
  14. 如权利要求13所述的用途，其特征在于，所述的药物通过雾化吸入治疗，从而达到阻止急性肺损伤炎症因子的释放、减轻肺泡内高蛋白液的渗入和肺泡上皮细胞损伤。
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