WO2022237247A1 - 细胞微颗粒在治疗呼吸道病毒性肺炎上的应用 - Google Patents

细胞微颗粒在治疗呼吸道病毒性肺炎上的应用 Download PDF

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WO2022237247A1
WO2022237247A1 PCT/CN2022/075503 CN2022075503W WO2022237247A1 WO 2022237247 A1 WO2022237247 A1 WO 2022237247A1 CN 2022075503 W CN2022075503 W CN 2022075503W WO 2022237247 A1 WO2022237247 A1 WO 2022237247A1
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黄波
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中国医学科学院基础医学研究所
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    • C12N5/0602—Vertebrate cells
    • C12N5/0693—Tumour cells; Cancer cells
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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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
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    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
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    • A61K9/00—Medicinal preparations characterised by special physical form
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    • A61K9/007—Pulmonary tract; Aromatherapy
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    • A—HUMAN NECESSITIES
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    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5063—Compounds of unknown constitution, e.g. material from plants or animals
    • A61K9/5068—Cell membranes or bacterial membranes enclosing drugs
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    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • This application relates to biological, medical and clinical fields. Specifically, it relates to the use of cell microparticles in the treatment of novel coronavirus pneumonia infection.
  • Viral pneumonia can break out or spread. Viral pneumonia can occur at any time of the year, but is more common in winter and spring. Initially, viral pneumonia is mainly upper respiratory tract virus infection, as the virus spreads downward to the lungs to cause pneumonia.
  • Viral pneumonia can be transmitted by droplets.
  • the clinical manifestations are generally mild, mainly with symptoms similar to respiratory diseases such as headache, fatigue, fever, and cough.
  • Respiratory tract infection is one of the leading causes of death worldwide, especially in severe pneumonia patients with high mortality and severe sequelae.
  • viruses such as influenza virus and coronavirus are the main pathogens causing regional outbreaks of severe pneumonia, which are highly contagious and have a high fatality rate.
  • COVID-19 is an acute respiratory infectious disease caused by 2019 novel coronavirus (SARS-CoV-2) infection.
  • 2019 novel coronavirus SARS-CoV-2
  • the 2019 novel coronavirus is a new strain of coronavirus that has never been found in humans before, and the main route of transmission is respiratory droplet transmission and contact transmission.
  • Novel coronavirus pneumonia mainly manifests as fever, dry cough, and fatigue.
  • a small number of patients are accompanied by upper respiratory and gastrointestinal symptoms such as nasal congestion, runny nose, and diarrhea.
  • Severe patients often develop dyspnea within a week, and severe cases rapidly progress to acute respiratory distress syndrome, septic shock, coagulation dysfunction, and multiple organ failure, which eventually lead to the death of the patient.
  • the novel coronavirus is highly contagious and has a high fatality rate.
  • the present application provides a tumor cell-derived microparticle, which uses cell vesicles derived from apoptotic tumor cells and expresses spike protein-binding receptors on them as a targeting agent, which is more conducive to reaching the treatment site for pneumonia.
  • the cell is composed of the content of the cell wrapped by the cell membrane, and the cell membrane is composed of protein molecules embedded in the middle of the phospholipid bilayer. maintain.
  • the size of the vesicles is about 100 to 1000 nanometers, "cellular vesicles" as described in this application.
  • the above-mentioned cell vesicle is used as a carrier, and the targeting agent is expressed on it, which is more conducive to targeting the therapeutic target.
  • Such vesicles cannot enter normal tissues (the permeability is about 5 to 10nm), and will not cause damage to normal tissues, thus avoiding the side effects of exogenous carriers such as nanomaterials on the body.
  • the cell vesicles are derived from tumor cells, especially from the same type of tumor cells as the site to be treated.
  • the cellular vesicles can readily contact the cell membrane of the site to be treated in the patient.
  • the patient's site to be treated is the lungs.
  • the tumor cells are lung cancer cells; thus the cellular exosomes are derived from lung cancer cells.
  • the tumor cell type used to prepare the cell vesicles and the cell type at the site where the cell vesicles are to be administered may be different.
  • the cell vesicles of the present application are prepared from colon cancer cells, yet the cell vesicles allow administration to the lung.
  • the tumor cells used to prepare the cell vesicles themselves contain oxidized cholesterol, eg, hydroxylated cholesterol.
  • the tumor cells used to make the cell vesicles contain 25-hydroxylated cholesterol.
  • tumor cell lines There are many human tumor cell lines known and available in the art, and the content of oxidized cholesterol in them varies. In some embodiments, tumor cell lines rich in oxidized cholesterol (eg, hydroxylated cholesterol) are allowed to be selected as a source for the production of cellular vesicles.
  • oxidized cholesterol eg, hydroxylated cholesterol
  • the tumor cells are lung cancer cells, such as but not limited to: NCI-H196, NCI-H292, NCI-H460, NCI-H446, NCI-H1299, NCI-H1650, H1792, NCI-H3255, A427, A549 , 0225-02Sp, 2F7, 95-D, SPCA-1, LLC, Calu-1, Calu-3, L1022, PC9R, MSTO-211H, TKB-1.
  • the tumor cells are Calu-3 enriched in oxidized cholesterol.
  • the tumor cell is A549 rich in oxidized cholesterol.
  • the tumor cells are colon cancer cell lines.
  • the colon cancer cells are selected from: HCT116, HCT116/FU, HCT-8/FU, LoVo, LoVo ADR, SW480, SW620, CaCo-2, RKO-E6, RKO-AS45-1, FET, HT55, HT115, HT -29, COLO 205, KM12, CL-40, KM12-SM, COLO320DM, NCI-H508, SW1417, COLO394, WiDr.
  • the tumor cells are Caco-2 enriched in oxidized cholesterol.
  • the targeting agent is a spike protein binding receptor expressed on the surface of the vesicle.
  • the spike protein-binding receptor expressed on the cell vesicle can be expressed naturally or recombinantly.
  • the Spike protein is a pneumovirus Spike protein (S; Spike).
  • the pneumovirus is selected from: SARS-CoV, SARS-CoV-2, or variants thereof.
  • the spike protein binding receptor is angiotensin converting enzyme 2 or a binding fragment thereof.
  • ACE2 is human ACE2.
  • the term encompasses naturally occurring human ACE2 or naturally occurring variants thereof, as well as artificially expressed human ACE2 or variants thereof, such as recombinant in vitro expressed human ACE2 or variants thereof.
  • binding fragment of angiotensin-converting enzyme 2 refers to a fragment of angiotensin-converting enzyme 2, as long as it still retains the ability to specifically bind to the spike protein, it falls within the scope of this application.
  • the angiotensin-converting enzyme 2 or its binding fragments in the present application can be expressed naturally, recombinantly expressed, or genetically modified.
  • the tumor cells are selected from: lung cancer cells, colon cancer cells.
  • the present application provides a method for preparing microparticles derived from tumor cells, and cell vesicles are obtained by apoptotic tumor cells by any feasible method.
  • a method for preparing tumor cell-derived microparticles comprising the steps of:
  • step 2) making the tumor cells in step 1) apoptotic
  • the cells are apoptotic by any method known in the art.
  • Non-limiting examples include ultraviolet radiation, X-ray radiation, or chemotherapy drugs such as dexamethasone.
  • an apoptotic method that does not introduce foreign substances into the tumor cells can be used, so radiation (for example, ultraviolet irradiation, X-ray irradiation) is preferred.
  • the duration and intensity of radiation can be determined by skilled personnel according to routine operations.
  • an ultracentrifuge can be used to separate at low temperature (or room temperature).
  • the cell vesicles are collected by a centrifuge at a low temperature (such as about 4° C.) with a centrifugal force of 100 to 100,000 g.
  • the cell vesicles released by the apoptotic tumor cells are collected by centrifugation, and the average particle size of the cell vesicles is 200nm to 800nm, preferably 300nm, 400nm, 500nm, 600nm, 700nm, 800nm ⁇ 10%.
  • a method for preparing tumor cell-derived microparticles comprising the steps of:
  • human tumor cells expressing human ACE2 or a binding fragment thereof, wherein the human tumor cells are lung cancer cells or colon cancer cells;
  • step 2) making the tumor cells in step 1) undergo apoptosis
  • the second product was centrifuged at 12000 to 15000 g for 40 to 80 minutes at 2 to 8° C. to collect the released cell vesicles.
  • the cell vesicles released by the apoptotic tumor cells are collected by the following method, comprising the steps of:
  • the present application provides a tumor cell-derived microparticle prepared by the aforementioned method.
  • the present application provides a pharmaceutical composition comprising the tumor cell-derived microparticles according to the present application.
  • the pharmaceutical composition comprises the cell vesicles or microparticles derived from tumor cells.
  • the collected microparticles can be made into pharmaceutical compositions, especially atomized formulations or spray formulations, according to conventional methods.
  • the average particle diameter of the pharmaceutical composition formed by the tumor cell-derived microparticles is 100 to 1000 nanometers, non-limiting examples that can be mentioned are 100, 150, 200, 250, 300 nanometers , 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 ⁇ 10% nm.
  • the pharmaceutical composition provided in this application can be administered according to conventional clinical treatment methods.
  • for pneumonia it can be administered directly through nasal drops, sprays, or perfusion into the lungs; the dosage can be used according to the dosage determined by medical workers.
  • microparticles, cell vesicles, and pharmaceutical compositions provided herein are prepared into dosage forms for pulmonary administration.
  • inhalable powders for pulmonary administration can be produced by conventional techniques such as jet milling, spray drying, solvent precipitation, supercritical fluid condensation, and the like.
  • dry powder inhalers dry powder inhalers, DPI
  • metered dose inhalers metered dose inhalers
  • GSK GSK
  • Astra TeurbohalerTM
  • a suitable carrier such as mannitol , sucrose or lactose
  • Solution formulations with liposomes can also be delivered to the lungs using an ultrasonic nebulizer.
  • a microparticle derived from A549 cells which can be used as a treatment for patients infected with novel coronavirus pneumonia.
  • Angiotensin-converting enzyme 2 (ACE2) present on the surface of microparticles, is a receptor that can bind to SARS-CoV-2 surface proteins and mediate viral entry. Therefore, MPs can adsorb the SARS-CoV-2 virus and limit the spread of the virus in the body. Virus-carrying microparticles will be efficiently taken up by macrophages and transported to lysosomes for degradation, thereby achieving effective treatment of new coronavirus pneumonia.
  • microparticle refers to a vesicle-like structure that eukaryotic cells peel off from the cell membrane surface during activation or apoptosis, with an average particle size of 100 to 1000 nm. Microparticles are considered as a biological information carrier, which mediates the transfer and exchange of biological information substances between different types of cells. Due to the characteristics of high biocompatibility, low immunogenicity, and targeting, it can be used as a drug carrier. Tumor cell-derived microparticles loaded with anti-tumor drugs have good anti-tumor effects and have been used in clinical practice.
  • the animal experiment dose of the microparticle preparation is 5 ⁇ 10 6 /50 ⁇ l, administered once a day for 5 days.
  • a skilled person can determine the unit dose for human subjects based on animal experiments.
  • Figure 1 shows that ACE2 is expressed in microparticle MPs derived from A549 cells, and Western blot analysis proves that ACE2 is expressed in MPs.
  • FIG. 2A and Figure 2B show that SARS-CoV-2 can be adsorbed by MPs.
  • the solution was filtered through a 0.1Mp-type filter, and real-time PCR analysis showed that viral RNA could be detected in incubated MPs, but not in unincubated MPs (Fig. 2A). This was further confirmed by immunofluorescent staining, showing that the viral S protein binds to ACE2 on MPs (Fig. 2B).
  • Figure 3 shows that MPs adsorb virions and transport them to alveolar macrophages.
  • In vitro incubation of alveolar macrophages with virus-adsorbed MPs showed that macrophages could uptake virus-containing MPs within 10 min, whereas isolated primary type II alveolar epithelial cells had difficulty uptake of MPs.
  • Figure 4 shows that SARS-CoV-2 virus adsorbed by MPs is inactivated in alveolar macrophages. After incubation of SARS-CoV-2, SARS-CoV-2/MPs with alveolar macrophages for 0.5, 1 and 4 hours, SARS-CoV-2 could replicate in alveolar macrophages, and SARS-CoV bound to MPs- 2 In alveolar macrophages with reduced load, probe 1 targets viral positive sequences to assess viral distribution (green), and probe 2 targets viral negative sequences to indicate viral replication (red) (according to conventional design principles, technicians able to design their own probes).
  • Figure 5A shows confocal microscopy images, bar 5 ⁇ m.
  • FIGS 5B to 5D show that cholesterol 25-hydroxylase in A549-ACE2-OE cells after irradiation was significantly up-regulated, and the content of 25-hydroxycholesterol in the extracted microparticles was significantly increased.
  • 293T means human renal epithelial 293 cell line (a cell line with low content of hydroxylated cholesterol);
  • 293T-UV means 293T cells after 1 h of ultraviolet irradiation;
  • A549-OE means A549 ACE2 overexpression cells;
  • 293-MPs represents microparticles derived from 293T cells;
  • AO-MPs represents microparticles derived from A549-ACE2 overexpression cells.
  • FIG. 5E shows that knocking out cholesterol-25-hydroxylase (abbreviated as CH25H), the effect of microparticles on up-regulating endosomal pH disappears.
  • Ctrl represents only PBS group;
  • SGCtrl represents the control microparticle group derived from A549;
  • SG1 represents the microparticle group derived from A549-CH25H knockout cells;
  • SG2 represents the microparticle group derived from A549-CH25H knockout cells.
  • FIG. 6A and Figure 6B show that MPs enhance the degradation of SARS-CoV-2 virus by reducing the pH of lysosomes.
  • Treatment of alveolar macrophages with MPs resulted in a decrease in lysosomal pH (Fig. 6A), and lysosomes isolated from MPs-treated macrophages inactivated the virus more efficiently than lysosomes isolated from control macrophages (Fig. 6B ).
  • Figure 7A to Figure 7B show the effect of MPs in treating SARS-CoV-2 virus infection in vivo.
  • Mice were injected with MPs 5 days after SARS-CoV-2 infection, and continued for 5 days.
  • H&E staining showed that the infiltration of inflammatory cells around the bronchi and perivascular of the mice in the treatment group was reduced, and the pathological damage of lung tissue was alleviated (Fig. 7A).
  • RNA Scope showed a reduction in viral load in the lung (Fig. 7B).
  • Ctrl control
  • Mock PBS treatment group.
  • Figure 8A shows the levels of TNF- ⁇ , IL-1 ⁇ , and IL-6 inflammatory factors in macrophages ingesting MPs carrying SARS-CoV-2 in vitro compared with those ingesting SARS-CoV-2 alone.
  • Figure 8B shows the levels of TNF- ⁇ , IL-1 ⁇ , and IL-6 inflammatory factors in the lung tissue of mice treated with MPs. Note: Ctrl: control; Mock: PBS treatment group.
  • Raw264.7 mouse macrophage cell line and A549 human lung adenocarcinoma cell line were purchased from CCTCC, China Center for Type Collection;
  • mice Female ICR, hACE2 transgenic mice, 6 to 8 weeks old, were purchased from the Medical Experimental Animal Center of the Chinese Academy of Medical Sciences (Beijing), and studies on mice without virus infection were approved by the Animal Protection and Use Committee of the Chinese Academy of Medical Sciences.
  • Example 1 Expression of ACE2 in microparticle MPs derived from A549 cells
  • the coding sequence of human ACE2 was amplified and inserted into the plasmid pLV-EF1 ⁇ -IRES-Puro, and expressed transiently in 293T cells to obtain the virus containing human ACE2 gene.
  • the lentivirus containing human ACE2 was transduced into A549 cells, and then the cells with high expression of ACE2 clone were obtained by screening with 1 ⁇ g/ml puromycin, that is, A549-ACE2-OE.
  • knockout RNA was designed for the known human ACE2 gene sequence in the database.
  • the RNA was cloned into the pSpCas9(BB)-2A-GFP vector plasmid and transfected into cells. After 48 hours, GFP-positive cells were sorted by flow cytometry using a BD Biosciences FACSAria III.
  • Candidate knockout cells were verified by Western blotting or immunofluorescence to obtain A549-ACE2-SG (A549 cells with ACE2 knockout).
  • A549, A549-ACE2-OE, and A549-ACE2-SG were irradiated with 300J/m 2 ultraviolet light for 1.5 hours, and the supernatant was collected after 18 hours.
  • the supernatant was centrifuged at 1000 ⁇ g for 10 min to remove cells, then at 14000 ⁇ g for 2 min to remove debris. Then, take the supernatant and centrifuge at 14000 ⁇ g for 60 min at 4°C to obtain MPs particles. MPs were washed 3 times, suspended in culture medium, and carried out subsequent experiments.
  • A549 cells and each MPs were lysed in lysis buffer and treated with sonication. Protein concentration was determined by BCA kit. Then, run the protein on an SDS-PAGE gel and transfer it to a nitrocellulose membrane. Nitrocellulose membrane was blocked with 5% bovine serum albumin and detected overnight with antibody to detect the expression of ACE2 in MPs. A549 cells were used as positive control.
  • Embodiment 2.SARSCoV-2 virus can be adsorbed by MPs
  • MPs obtained from A549-ACE2-OE were incubated with SARSCoV-2 virus at 37°C for 30 min, and then filtered with a 0.1 ⁇ m filter membrane.
  • the filter selectively allows virus particles to pass through, but blocks MPs.
  • the SARSCoV-2 virus that was not incubated with MPs was used as a control group, which was also filtered through the filter membrane, and the viral load on the filter membranes of different groups was detected by real-time fluorescent quantitative PCR.
  • MPs (5 ⁇ 10 5 ) were incubated with recombinant SARS-CoV-2 spike protein (0.1 ⁇ g), fixed and stained with anti-ACE2 protein (red) and anti-spike protein (green) antibodies. Fluorescence was measured by super-resolution structured illumination microscopy, scale bar 2 ⁇ m.
  • mice Isolation of primary alveolar macrophages from mouse bronchoalveolar lavage fluid.
  • Mice were anesthetized immediately before lavage, and trachea were dissected.
  • Lungs were lavaged 5 times with 1ml of PBS, and the lavage solution was centrifuged at 600 ⁇ g for 5 minutes at 4°C.
  • the collected cells were suspended in RPMI1640 complete medium, cultured on a culture plate for 2 hours, and then washed gently with PBS to remove unlabeled cells. parietal cells.
  • mice Primary alveolar epithelial cells were isolated from hACE2 mice, and the mice were perfused via the right ventricle with 10 ml of cold PBS. Fill the lung tissue with 2ml racemase and low gel temperature agarose gel, then incubate the lung tissue with 2ml racemase at 37°C for 20min. Lung tissue was then ground and the slurry was filtered through 70 and 40 ⁇ m nylon mesh. Cell suspension was used biotin-labeled antibody and magnetic beads to exclude leukocytes, monocytes/macrophages, NK cells, neutrophils, endothelial cells and erythroid cells.
  • MPs (5 ⁇ 10 5 ) and SARS-CoV-2 (5 ⁇ 10 4 TCID 50 ) were incubated at 37°C for 30 min, and then alveolar macrophages were added for 30 min, 1 h and 4 h. MPs unincubated with SARS-CoV-2 were used as the control group.
  • Cells were fixed in 4% paraformaldehyde, incubated with hydrogen peroxide for 10 min at room temperature, and RNA in situ analysis was performed using the RNAScope kit.
  • Probe 1 targets the positive sequence of the virus to assess virus distribution (green)
  • probe 2 targets the negative sequence of the virus to indicate viral replication (red)
  • scale bar 5 ⁇ m accordinging to conventional design principles, technicians can design probe 1 and probe needle 2).
  • SARS-CoV-2 After incubation of SARS-CoV-2, SARS-CoV-2/MPs with alveolar macrophages for 0.5, 1 and 4 hours, SARS-CoV-2 alone could replicate in alveolar macrophages, and SARS-CoV bound to MPs The -2 load was reduced in alveolar macrophages (Fig. 4).
  • Macrophages were treated with microparticles for 30 minutes in advance, and then added with pHrodo TM Red dextran to label for 10 minutes, and photographed with a confocal microscope. Scale bar 5 ⁇ m.
  • A549-ACE2-OE cells were plated in a six-well plate (5x10 5 ) and irradiated with 300J/m2 ultraviolet light for 1 hour, and RNA was collected after 18 hours, and the content of CH25H was detected by qPCR. At the same time, microparticles were extracted by this method, and the content of 25-hydroxycholesterol (25HC) in MPs was detected by mass spectrometry.
  • the CH25H of A549 cells was knocked out using the CRISPR-Cas9 system, MPs were extracted, macrophages were treated for 30 minutes, and then pHrodo TM Red dextran was added for labeling for 10 minutes, and photographed with a confocal microscope (Figure 5A).
  • microparticles could increase the pH value of endosomes and hinder virus escape to cytoplasmic replication.
  • pH of macrophage endosomes increased.
  • Oxidized cholesterol carried by microparticles affects the pH of endosomes.
  • Figure 5B, Figure 5C, and Figure 5D show that cholesterol 25-hydroxylase in A549-ACE2-OE cells after irradiation was significantly up-regulated, and the content of 25-hydroxycholesterol in the extracted microparticles was also significantly increased.
  • Figure 5E shows that knocking out CH25H, the effect of microparticles on upregulating endosomal pH disappeared.
  • Alveolar macrophages were pretreated with MPs obtained from A549-ACE2-OE for 30 minutes, alveolar macrophages not treated with MPs were used as a control group, stained with LysoSensor TM yellow/blue DND-160, and detected with a microplate reader pH value, lysosome pH measuring instrument LysoSensor TM yellow/blue DND-160 is used to measure the pH of macrophage lysosomes, and the results show that the pH of living cells depends on dual excitation spectra.
  • Lysosomes were isolated and purified, and the adherent cells were digested with trypsin and washed with ice-cold PBS. The cell pellet was resuspended and broken up in a pulper. The cell homogenate was centrifuged at 1000 ⁇ g for 10 minutes, and the supernatant was centrifuged at 20000 ⁇ g for 20 minutes to make lysosome particles and other organelles. By establishing a density gradient and centrifuging at 150,000 x g for 4 h in a SW50.1 rotor, the highest (lowest density) band was removed and diluted in PBS. After washing, the lysosomes were separated and purified by centrifugation at 20000 ⁇ g for 20 min.
  • Lysosomes isolated from macrophages treated with MPs and lysosomes isolated from control macrophages were incubated with SARS-CoV-2 at 37°C for 30 min, and then added virus E6 to infect for 48 h. Cells were stained with anti-NP antibody. Scale bar 5 ⁇ m.
  • Example 7 Effect of MPs in vivo treatment of SARS-CoV-2 virus infection
  • RNA in situ analysis was performed using the RNAScope kit. Probe 1 was directed against the positive sequence of the virus to evaluate the distribution of the virus (green), and probe 2 was directed against the negative sequence of the virus to indicate the replication of the virus (red).
  • the scale bar is 5 ⁇ m.
  • Example 8 Macrophages do not cause an inflammatory response when eliminating SARS-CoV-2 adsorbed by MPs
  • Macrophages (1x10 5 ) were added with SARS-CoV-2 (1x10 5 TCID50) for 24 hours, RNA was extracted by TRIZOL method, and the levels of TNF- ⁇ , IL-1 ⁇ , and IL-6 were detected by qPCR.
  • Figure 8A shows that the ingestion of MPs carrying SARS-CoV-2 by macrophages in vitro significantly decreased the levels of TNF- ⁇ , IL-1 ⁇ , and IL-6 inflammatory factors compared with the simple ingestion of SARS-CoV-2.
  • Figure 8B shows that the levels of TNF- ⁇ , IL-1 ⁇ , and IL-6 inflammatory factors in the lung tissue of mice treated with MPs were significantly down-regulated.
  • the A549 cell-derived microparticles (MPs) provided by this application are used for the treatment of novel coronavirus infection, using its characteristics of adsorbing SARS-CoV-2 virus and enhancing the ability of alveolar macrophages to scavenge novel coronavirus.
  • MPs cell-derived microparticles
  • the ACE2 existing on the surface of the microparticles can bind to the S protein on the surface of SARS-CoV-2 and adsorb virus particles, limiting the further spread of the virus in the body.
  • Virus-carrying microparticles will be efficiently taken up by macrophages and transported to lysosomes, and MPs can enhance the ability to clear SARS-CoV-2 by adjusting the pH value of macrophage lysosomes, so as to realize the clinical treatment of new coronavirus pneumonia. Treatment can effectively reduce the mortality rate of patients with new coronary pneumonia, and has a good clinical application prospect.
  • microparticles of this application are derived from cells, and have the characteristics of high biocompatibility, low immunogenicity, and targeting, so they are safe and have no toxic or side effects.

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Abstract

一种细胞微颗粒在治疗呼吸道病毒性肺炎上的应用。一种肿瘤细胞来源的微颗粒(MPs)，其包含凋亡的肿瘤细胞所释放的细胞囊泡，所述细胞囊泡在其表面表达刺突蛋白结合受体。MPs具有较高的生物安全性，发现微颗粒表面的受体具有吸附病毒的能力，并且巨噬细胞能高效地摄取MPs，可用于治疗病毒性肺炎。

Description

细胞微颗粒在治疗呼吸道病毒性肺炎上的应用 技术领域
本申请涉及生物、医学、临床领域。具体而言，涉及细胞微颗粒在治疗新型冠状病毒肺炎感染中的用途。
背景技术
病毒性肺炎可爆发，也可散发流行。病毒性肺炎可在一年中的任何时候发生，但多见于冬春季节。最初，病毒性肺炎以上呼吸道病毒感染为主，随着病毒向下蔓延至肺部引起肺炎。
病毒性肺炎可通过飞沫传染。临床表现一般较轻，以头痛、乏力、发热、咳嗽等呼吸道疾病相似症状为主。呼吸道感染是全球最主要的死亡原因之一，尤其是重症肺炎患者出现高死亡率和严重的后遗症。目前，流感病毒、冠状病毒等病毒是导致区域爆发性重症肺炎的主要病原体，具有强传染性和高病死率。
新型冠状病毒肺炎COVID-19是由2019新型冠状病毒(SARS-CoV-2)感染引起的急性呼吸道传染病。
2019新型冠状病毒是以前从未在人体中发现的冠状病毒新毒株，主要的传播途径还是呼吸道飞沫传播和接触传播。新型冠状病毒肺炎主要表现为发热、干咳、乏力，少数患者伴有鼻塞、流涕、腹泻等上呼吸道和消化道症状。重症患者多在一周左右出现呼吸困难，严重者快速进展为急性呼吸窘迫综合征、脓毒症休克、出凝血功能障碍及多器官功能衰竭等，最终导致患者死亡。新型冠状病毒具有强传染性和高病死率。
新型冠状病毒感染的肺炎疫情暴发后，尽管各国在药物开发方面做出了很大努力，但到目前为止，仍然没有有效治疗SARS-CoV-2感染的药物。对于新型冠状病毒肺炎的防治，疫苗研发取得了很大的进展，中国已经附条件上市的新冠疫苗已经达到4个，其中三个灭活疫苗，一个腺病毒载体疫苗。虽然疫苗可以极大地防止病毒传播，但是对于已经感染病毒的患者仍然需要探索更为有效的治疗方案。目前针对新型冠状病毒肺炎的药物研发主要是筛选针对病毒复制和包装的小 分子化合物，然而许多小分子化合物面临着不确定性和安全性的挑战。尽管许多药物在体外展现出了良好的抗冠状病毒活性，但往往不具有人体内应用的价值。有一些药物已经用于临床抗病毒治疗，但是这些药物对于新型冠状病毒都没有特异性，其副作用也不容忽视。
因此，有必要探索新型冠状病毒肺炎患者的非常规治疗策略。
发明内容
本申请提供了一种肿瘤细胞来源的微颗粒，以源自凋亡肿瘤细胞的细胞囊泡，其上表达刺突蛋白结合受体而作为靶向剂，更利于达到肺炎治疗部位。
技术人员知晓，细胞是由细胞膜包裹细胞的内容物构成，而细胞膜是由磷脂双分子层中间镶嵌蛋白质分子所组成，细胞的球状结构通过称为细胞骨架的蛋白纤维丝所形成的牵拉力得以维持。当细胞受到外来信号(例如：化疗药、紫外线、辐射等)刺激而发生凋亡时，蛋白纤维丝断裂或失去附着，牵拉力消失，使得局部细胞膜结构向外膨胀、突出并包裹细胞内容物以囊泡形式释放，囊泡的大小约为100至1000纳米，本申请所述的“细胞囊泡”。
采用了上述细胞囊泡作为载体，在其上表达靶向剂，更利于靶向治疗靶点。这种囊泡不能进入正常组织(其通透性约为5至10nm)，对正常组织不会产生损伤，也就避免了使用纳米材料等外源载体对机体的副作用。
在一些实施方案中，所述细胞囊泡源自肿瘤细胞，尤其是源自与所要治疗的部位同种类型的肿瘤细胞。在一些实施方案中，所述细胞囊泡可以很容易地与患者体内的待治疗部位的细胞膜接触。在一些具体的实施方案中，患者的待治疗部位是肺。在一些实施方案中，肿瘤细胞是肺癌细胞；因而细胞囊泡源自肺癌细胞。
在另一些实施方案中，制备细胞囊泡所用的肿瘤细胞类型和细胞囊泡待施用部位的细胞类型，可以是不同的。作为一个示例，本申请的细胞囊泡制备自结肠癌细胞，然而所述细胞囊泡允许施用至肺。
在具体的实施方案中，制备细胞囊泡所用的肿瘤细胞，其自身包含氧化型胆固醇，例如包含羟基化修饰的胆固醇。作为一个示例，制 备细胞囊泡所用的肿瘤细胞包含25-羟基化胆固醇。
本领域中公知且可获得的人肿瘤细胞系众多，所含氧化型胆固醇的含量存在差异。在一些实施方案中，允许选择富含氧化型胆固醇(如羟基化胆固醇)的肿瘤细胞系作为制备细胞囊泡的来源。
在一些实施方案中，肿瘤细胞是肺癌细胞，例如但不限于：NCI-H196、NCI-H292、NCI-H460、NCI-H446、NCI-H1299、NCI-H1650、H1792、NCI-H3255、A427、A549、0225-02Sp、2F7、95-D、SPCA-1、LLC、Calu-1、Calu-3、L1022、PC9R、MSTO-211H、TKB-1。在一个具体的实施方案中，肿瘤细胞是富含氧化型胆固醇的Calu-3。在一个具体的实施方案中，肿瘤细胞是富含氧化型胆固醇的A549。
在一些实施方案中，肿瘤细胞是结肠癌细胞系。所述结肠癌细胞选自：HCT116、HCT116/FU、HCT-8/FU、LoVo、LoVo ADR、SW480、SW620、CaCo-2、RKO-E6、RKO-AS45-1、FET、HT55、HT115、HT-29、COLO 205、KM12、CL-40、KM12-SM、COLO320DM、NCI-H508、SW1417、COLO394、WiDr。在一个具体的实施方案中，肿瘤细胞是富含氧化型胆固醇的Caco-2。
在一些实施方案中，所述靶向剂是在囊泡表面表达的刺突蛋白结合受体。
在一些实施方案中，细胞囊泡上表达的刺突蛋白结合受体，可以是天然表达的，也可以是重组表达的。
在一些实施方案中，刺突蛋白是肺炎病毒刺突蛋白(S；Spike)。
在一些实施方案中，所述肺炎病毒选自：SARS-CoV、SARS-CoV-2、或其变体。
在一些具体的实施方案中，所述刺突蛋白结合受体是血管紧张素转换酶2或其结合片段。
在一些具体的实施方案中，ACE2是人ACE2。该术语涵盖天然存在的人ACE2或其天然存在的变体，也包括人为表达的人ACE2或其变体，如重组体外表达的人ACE2或其变体。
本申请所述“血管紧张素转换酶2的结合片段”是指血管紧张素转换酶2的片段，只要其仍保留特异性结合刺突蛋白的能力，均属于本申请的范畴。本申请中的血管紧张素转换酶2或其结合片段可以是 天然表达的、也可以是重组表达的、或者经基因改造的。
在一些实施方案中，所述肿瘤细胞选自：肺癌细胞、结肠癌细胞。
本申请提供了一种制备肿瘤细胞来源的微颗粒的方法，通过任何可行的方法使肿瘤细胞凋亡而得到细胞囊泡。
在一些具体的实施方案中，提供了一种制备肿瘤细胞来源的微颗粒的方法，包括步骤：
1)提供表达刺突蛋白结合受体的肿瘤细胞；
2)使步骤1)的所述肿瘤细胞凋亡；
3)收集凋亡的肿瘤细胞所释放的细胞囊泡。
在一些具体的实施方案中，通过本领域任何公知的方法使细胞凋亡。作为非限制性示例，包括紫外线照射、X射线照射、或化疗药物(如地塞米松)。在本申请的方法中，可以采用不向肿瘤细胞引入外源物质的凋亡方法，因而优选辐射的方式(例如，紫外线照射、X射线照射)。在一些具体的实施方案中，辐射的时间长短和辐射强度，是技术人员根据常规操作能够确定的。
本申请的具体实施方案中，优选使用紫外线或化疗药物诱导肿瘤细胞凋亡，对于细胞囊泡的收集可使用超速离心机在低温条件下(或室温)条件下进行分离。优选的，通过离心机在低温条件下(如4℃左右)，以100至100000g的离心力，收集细胞囊泡。
在一些具体的实施方案中，通过离心收集凋亡的肿瘤细胞所释放的细胞囊泡，所述细胞囊泡的平均粒径为200nm至800nm，优选300nm、400nm、500nm、600nm、700nm、800nm±10％。
在一些具体的实施方案中，提供了一种制备肿瘤细胞来源的微颗粒的方法，包括步骤：
1)提供表达人ACE2或其结合片段的人肿瘤细胞，所述人肿瘤细胞是肺癌细胞或结肠癌细胞；
2)使步骤1)的所述肿瘤细胞发生凋亡；
3)通过以下方式收集凋亡的肿瘤细胞所释放的细胞囊泡，包括步骤：
在2至8℃，以800至1000g对步骤2)所得凋亡的肿瘤细胞离心5至15分钟，以去除完整细胞，得到第一产物；
在2至8℃，以12000至15000g对第一产物离心1至3分钟，以去除细胞碎片，得到第二产物；
在2至8℃，以12000至15000g对第二产物离心40至80分钟，以收集释放的细胞囊泡。
在一些具体的实施方案中，通过以下方式收集凋亡的肿瘤细胞所释放的细胞囊泡，包括步骤：
在2至8℃，以1000g对所得凋亡的肿瘤细胞离心10分钟，以去除完整细胞，得到第一产物；
在2至8℃，以14000g对第一产物离心2分钟，以去除细胞碎片，得到第二产物；
在2至8℃，以14000g对第二产物离心60分钟，以收集释放的细胞囊泡；按此步骤收集的细胞囊泡使其平均粒径落入100nm至1000nm的范畴，尤其是200至800nm。
本申请提供了一种通过前述方法制备的肿瘤细胞来源的微颗粒。
本申请提供了一种药物组合物，其包含根据本申请的肿瘤细胞来源的微颗粒。
根据本申请药物组合物的优选方案，该药物组合物包含所述细胞囊泡、或肿瘤细胞来源的微颗粒。
对于所收集到的微颗粒，可以按照常规方法制成药物组合物，尤其是雾化制剂或者喷剂制剂。
作为本申请的优选方案，由所述肿瘤细胞来源的微颗粒所形成的药物组合物的平均粒径为100至1000纳米，可以提及的非限制性示例是100、150、200、250、300、350、400、450、500、550、600、650、700、750、800、850、900、950、1000±10％nm。
本申请提供的药物组合物可以按照常规临床治疗方法给药，例如对于肺炎，可以直接滴鼻、喷雾、或灌注至肺部而给药；给药剂量可按照医疗工作者确定的剂量进行使用。
在具体的实施方案中，本申请提供的微颗粒、细胞囊泡、药物组合物制备成肺部施用的剂型。
作为一个示例，用于肺部施用可吸入的粉末可以通过常规的技术生产，如喷射制粉(jet milling)、喷雾干燥、溶剂沉淀、超临界流体 浓缩(supercritical fluid condensation)等。用干粉吸入器(dry powder inhalers，DPI)如基于NektarTM、Vectura(GyrohalerTM)和GSK(DiscusTM)或Astra(TurbohalerTM)等生产的计量吸入器(metered dose inhalers)，在合适的载体中(如甘露醇、蔗糖或乳糖)包含本申请的微颗粒、细胞囊泡或药物组合物，输送到末端肺泡表面。也可以使用超声波喷雾器，将有脂质体(或没有脂质体)的溶液制剂递送至肺部。
根据本申请的一些实施方案，提供了一种A549细胞来源的微颗粒，其可用作新型冠状病毒肺炎感染者的治疗手段。微颗粒表面存在的血管紧张素转换酶2(ACE2)是一种能与SARS-CoV-2表面蛋白结合并介导病毒进入的受体。因此MPs能吸附SARS-CoV-2病毒，限制病毒在体内传播。携带病毒的微颗粒会被巨噬细胞高效地摄取并输送至溶酶体，进行降解，从而实现对新型冠状病毒肺炎的有效治疗。
在本申请中，术语“微颗粒”(Microparticle，MP)是指真核细胞在活化或凋亡时从细胞膜表面剥落下来的囊泡状结构，平均粒径在100至1000nm。微颗粒被认为是一种生物信息载体，介导了生物信息物质在不同类型细胞间的传递和交换。由于生物相容性高、免疫原性低、靶向性等特点可用做药物的载体，肿瘤细胞来源微颗粒负载抗肿瘤药物，具有良好的抗肿瘤效果，并已应用于临床。
在一些实施方案中，所述微颗粒制剂动物实验剂量为5×10 6/50μl，每日一次，给药5天。技术人员可以根据动物实验，确定人受试者的单位剂量。
附图说明
图1显示了ACE2在A549细胞来源的微颗粒MPs中表达，Western印记分析证明了ACE2在MPs中表达。
图2A和图2B显示了SARS-CoV-2能够被MPs吸附。在SARS-CoV-2和AMPs孵育后，溶液经过0.1Mp型过滤器过滤，实时PCR分析表明，孵育MPs中可以检测到病毒RNA，未孵育MPs检测不到病毒RNA(图2A)。免疫荧光染色进一步证实，表明病毒S蛋白与MPs上的ACE2结合(图2B)。
图3显示了MPs吸附病毒粒子并输送到肺泡巨噬细胞。肺泡巨噬细胞与吸附病毒的MPs的体外孵育表明，巨噬细胞能在10分钟内摄取含病毒的MPs，而分离的原代II型肺泡上皮细胞很难摄取MPs。
图4显示了MPs吸附的SARS-CoV-2病毒在肺泡巨噬细胞中被灭活。在SARS-CoV-2、SARS-CoV-2/MPs与肺泡巨噬细胞孵育0.5、1和4小时后，SARS-CoV-2可以在肺泡巨噬细胞中复制，与MPs结合的SARS-CoV-2在肺泡巨噬细胞中载量降低，探针1针对病毒正向序列以评估病毒分布(绿色)，探针2针对病毒负向序列以指示病毒复制(红色)(根据常规设计原则，技术人员能够自行设计探针)。
图5A显示共聚焦显微镜图像，标尺5μm。
图5B至图5D显示辐照后的A549-ACE2-OE细胞胆固醇25羟化酶明显上调，提取的微颗粒25羟基胆固醇含量明显升高。注：293T表示人肾上皮293细胞系(一种羟基化胆固醇含量低的细胞系)；293T-UV表示紫外照射1h后的293T细胞；A549-OE表示A549 ACE2过表达细胞；A549-OE-UV表示紫外照射1h后的A549 ACE2过表达细胞；293-MPs表示293T细胞来源的微颗粒；AO-MPs表示A549-ACE2过表达细胞来源的微颗粒。
图5E显示敲除胆固醇-25-羟化酶(简称为CH25H)，微颗粒上调内小体pH的作用消失。Ctrl表示仅PBS的组；SGCtrl表示加A549来源的对照微颗粒组；SG1表示加A549-CH25H敲除细胞来源的微颗粒组；SG2表示加A549-CH25H敲除细胞来源的微颗粒组。
图6A和图6B显示了MPs通过降低溶酶体的pH增强对SARS-CoV-2病毒降解。肺泡巨噬细胞经MPs处理后导致溶酶体pH降低(图6A)，MPs处理的巨噬细胞分离的溶酶体比从对照巨噬细胞分离的溶酶体更有效地灭活病毒(图6B)。
图7A至图7B显示了MPs体内治疗SARS-CoV-2病毒感染的效果。SARS-CoV-2感染小鼠5天后鼻腔注射MPs，连续5天，H&E染色显示治疗组小鼠支气管周围和血管周围炎性细胞浸润减少，肺组织病理损害减轻(图7A)。与形态学的改善相一致，RNA Scope显示肺中的病毒载量减少(图7B)。Ctrl：对照；Mock：PBS处理组。
图8A显示体外巨噬细胞摄取携带SARS-CoV-2的MPs与单纯摄 取SARS-CoV-2相比，TNF-α、IL-1β、IL-6炎症因子的水平。
图8B显示经MPs治疗的小鼠肺组织TNF-α、IL-1β、IL-6炎症因子水平。注：Ctrl：对照；Mock：PBS处理组。
具体实施方式
实施例中使用的各种细胞系、药物及实验动物：
小鼠巨噬细胞系Raw264.7、A549人肺腺癌细胞系购自中国典型物保藏中心CCTCC；
雌性ICR，hACE2转基因小鼠，6至8周龄，购自中国医学科学院医学实验动物中心(北京)，没有病毒感染的小鼠研究得到了中国医学科学院动物保护与利用委员会的批准。
实施例1.A549细胞来源的微颗粒MPs中表达ACE2
1.实验步骤
将人ACE2编码序列扩增并插入质粒pLV-EF1α-IRES-Puro中，在293T细胞中瞬时表达，获得含人ACE2基因的病毒。将含人ACE2的慢病毒转导到A549细胞中，然后用1μg/ml嘌呤霉素筛选得到高表达ACE2克隆的细胞，即A549-ACE2-OE。
为构建稳定敲除ACE2细胞系，针对数据库中公知的人ACE2基因序列，设计敲除用RNA。将RNA克隆到pSpCas9(BB)-2A-GFP载体质粒并转染细胞。48小时后，使用BD Biosciences FACSAria III进行流式细胞术对GFP阳性细胞进行分类。候选敲除细胞通过Western印记或免疫荧光验证，获得A549-ACE2-SG(敲除ACE2的A549细胞)。
A549、A549-ACE2-OE、A549-ACE2-SG各自使用300J/m 2的紫外线照射1.5小时，18h小时后收集上清液。将上清液以1000×g离心10min去除细胞，然后以14000×g离心2min去除碎屑。然后，取上清液，以14000×g离心60min，4℃，制得各MPs颗粒。将MPs洗涤3次，悬浮于培养基中，进行后续实验。
A549细胞及各MPs分别在裂解缓冲液中裂解，超声处理。蛋白质浓度由BCA试剂盒测定。然后，在SDS-PAGE凝胶上运行该蛋白，并将其转移到硝酸纤维素膜上。硝酸纤维素膜用5％牛血清白蛋白封闭， 并用抗体检测过夜，检测MPs中ACE2的表达，A549细胞作为阳性对照。
2.实验结果
Western印记分析显示了MPs中ACE2表达，使用此方法获得的A549来源的微颗粒中存在ACE2(图1)。
实施例2.SARSCoV-2病毒能够被MPs所吸附
1.实验步骤
获自A549-ACE2-OE的MPs与SARSCoV-2病毒在37℃条件下孵育30min后，用0.1μm滤膜过滤。该过滤器可以选择性地允许病毒颗粒通过，但会阻止MPs通过。未孵育MPs的SARSCoV-2病毒作为对照组，同样经过滤膜过滤，用实时荧光定量PCR检测不同组滤膜上的病毒载量。
MPs(5×10 5)与重组SARS-CoV-2刺突蛋白(0.1μg)孵育，固定后用抗ACE2蛋白(红)和抗刺突蛋白(绿)抗体染色。用超高分辨率结构照明显微镜测定荧光，标尺2μm。
2.实验结果
在SARS-CoV-2和AMPs孵育后，Q-PCR分析表明，孵育MPs中可以检测到病毒RNA，未孵育MPs几乎检测不到病毒RNA(图2A)。免疫荧光染色结果证实，病毒S蛋白与MPs上的ACE2结合(图2B)。
实施例3.MPs吸附病毒粒子并输送到肺泡巨噬细胞
1.实验步骤：
从小鼠支气管肺泡灌洗液中分离出原代肺泡巨噬细胞。小鼠在灌洗前立即麻醉，并解剖气管。用1ml PBS对肺进行5次灌洗，灌洗液在4℃下600×g离心5min，收集的细胞在RPMI1640完全培养基中悬浮，在培养板上培养2h，然后用PBS温和洗涤去除未贴壁细胞。
从hACE2小鼠中分离出原代肺泡上皮细胞，小鼠经右心室灌注10毫升冷PBS。用2ml消旋酶和低凝胶温度琼脂糖凝胶填充肺组织，然后将肺组织与2ml消旋酶在37℃下孵育20min。然后研磨肺组织，并用70和40μm尼龙网过滤浆液。细胞悬液用生物素标记的抗体及磁珠 排除白细胞、单核/巨噬细胞、NK细胞、中性粒细胞、内皮细胞和红系细胞。
分离ICR小鼠的原代肺泡巨噬细胞和原代肺泡上皮细胞，用PKH67标记的A-OE-MPs处理10min、30min及2h后，在共聚焦显微镜下获取图像，比例尺5μm。
2.实验结果：
肺泡巨噬细胞与吸附病毒的MPs的体外孵育表明，巨噬细胞能在10分钟内摄取含病毒的MPs，而分离的原代II型肺泡上皮细胞2小时也很难摄取MPs(图3)。
实施例4.MPs吸附的SARS-CoV-2病毒在肺泡巨噬细胞中被灭活
1.实验步骤：
MPs(5×10 5)与SARS-CoV-2(5×10 4TCID 50)在37℃孵育30min，然后加入肺泡巨噬细胞处理30min，1h及4h。以SARS-CoV-2未孵育MPs作为对照组。细胞固定在4％多聚甲醛中，用过氧化氢在室温下孵育10min，使用RNAScope试剂盒进行RNA原位分析。探针1针对病毒正向序列以评估病毒分布(绿色)，探针2针对病毒负向序列以指示病毒复制(红色)，比例尺5μm(根据常规设计原则，技术人员能够自行设计探针1和探针2)。
2.实验结果：
在SARS-CoV-2、SARS-CoV-2/MPs与肺泡巨噬细胞孵育0.5、1和4小时后，单独SARS-CoV-2可以在肺泡巨噬细胞中复制，与MPs结合的SARS-CoV-2在肺泡巨噬细胞中载量降低(图4)。
实施例5.MPs通过增加内体pH阻止病毒逃匿
微颗粒提前处理巨噬细胞30min，然后加入pHrodo TM Red dextran标记10min，用共聚焦显微镜拍照。标尺5μm。
A549-ACE2-OE细胞铺于六孔板中(5x10 5)在经300J/m2的紫外线照射1小时，18h后收集RNA，进行qPCR检测CH25H的含量。同时利用该种方法提取微颗粒，利用质谱实验检测MPs中25-羟基胆固醇(25HC)的含量。利用CRISPR-Cas9系统将A549细胞CH25H敲 除，再提取MPs，处理巨噬细胞30min，然后加入pHrodo TM Red dextran标记10min，用共聚焦显微镜拍照(图5A)。
结果显示：微颗粒可以使内小体的pH值升高阻碍病毒逃逸到胞质复制。在经微颗粒处理后，巨噬细胞内小体pH值升高。微颗粒携带的氧化型胆固醇影响内小体的pH。图5B、图5C、图5D显示辐照后的A549-ACE2-OE细胞胆固醇25羟化酶明显上调，且提取的微颗粒25羟基胆固醇含量也明显升高。图5E显示敲除CH25H，微颗粒上调内小体pH的作用消失。
实施例6.MPs通过降低溶酶体的pH增强对SARS-CoV-2病毒降解
1.实验步骤
肺泡巨噬细胞用获自A549-ACE2-OE的MPs预处理30min，以未用MPs处理的肺泡巨噬细胞作为对照组，用LysoSensor TM黄/蓝DND-160型染色，用微量平板阅读器检测pH值，溶酶体pH测量仪LysoSensor TM黄/蓝DND-160用于测定巨噬细胞溶酶体的pH，结果显示活细胞的pH依赖于双激发光谱。
溶酶体分离纯化，贴壁细胞经胰酶消化后用冰PBS洗涤。将细胞沉淀物重新悬浮，放入浆机中破碎。细胞匀浆以1000×g离心10min，上清液以20000×g离心20min，制成溶酶体颗粒和其他细胞器。通过建立密度梯度，并在SW50.1转子中以150，000×g的速度离心4h，最高(最低密度)的条带被移除并在PBS中稀释。溶酶体洗涤后，以20000×g离心20min分离纯化。
将MPs处理的巨噬细胞分离的溶酶体和对照巨噬细胞分离的溶酶体分别与SARS-CoV-2在37℃孵育30min后加入病毒E6感染48h。用抗NP抗体对细胞进行染色。比例尺5μm。
2.实验结果
肺泡巨噬细胞经MPs处理后导致溶酶体pH降低(图6A)，MPs处理的巨噬细胞分离的溶酶体比从对照巨噬细胞分离的溶酶体的病毒量更低(图6B)。
实施例7.MPs体内治疗SARS-CoV-2病毒感染的效果
1.实验步骤
用SARS-CoV-2(1×10 5TCID 50)气管内感染hACE2小鼠，然后用对照组(PBS)或MPs(5×10 6)治疗。每日1次，连续5d(n＝5只/组)。治疗5d后处死小鼠，取肺组织固定进行HE染色。
肺组织固定后使用RNAScope试剂盒进行RNA原位分析，探针1针对病毒正向序列以评估病毒分布(绿色)，探针2针对病毒负向序列以指示病毒复制(红色)，比例尺5μm。
2.实验结果
H&E染色显示治疗组小鼠支气管周围和血管周围炎性细胞浸润减少，肺组织病理损害减轻(图7A)。与形态学的改善相一致，RNAScope显示肺中的病毒载量减少(图7B)。
实施例8.巨噬细胞在消灭MPs吸附的SARS-CoV-2时不引起炎症反应
巨噬细胞(1x10 5)加入SARS-CoV-2(1x10 5TCID50)24h，用TRIZOL法提取RNA，qPCR检测TNF-α、IL-1β、IL-6的水平。
气管内用SARS-CoV-2(1×10 5TCID50)感染hACE2小鼠，分为对照组(PBS)或MPs(5×10 6)治疗。每日1次，连续5天(n＝5只/组)。治疗5天后处死小鼠，TRIZOL法提取肺组织RNA，qPCR检测TNF-α、IL-1β、IL-6的水平。
图8A显示体外巨噬细胞摄取携带SARS-CoV-2的MPs与单纯摄取SARS-CoV-2相比，TNF-α、IL-1β、IL-6炎症因子水平明显下降。图8B显示经MPs治疗的小鼠肺组织TNF-α、IL-1β、IL-6炎症因子水平明显下调。
综上，本申请的方案具有以下效果：
1.本申请提供的A549细胞来源的微颗粒(MPs)用于治疗新型冠状病毒感染，利用其吸附SARS-CoV-2病毒的特性，并增强肺泡巨噬细胞对新冠病毒的清除能力，是一种新型的非常规治疗策略。
2.病毒感染后，使用气管内注射MPs，微颗粒表面存在的ACE2 可以与SARS-CoV-2表面S蛋白结合并吸附病毒颗粒，限制了病毒在体内进一步传播。携带病毒的微颗粒会被巨噬细胞高效地摄取并输送至溶酶体，MPs通过调节巨噬细胞溶酶体PH值增强清除SARS-CoV-2的能力，从而实现对新型冠状病毒肺炎的临床治疗，能够有效降低新冠患者的死亡率，有很好的临床应用前景。
3.本申请的微颗粒来源于细胞，具备生物相容性高、免疫原性低、靶向性等特点，因此安全性高、无毒副作用。

Claims (9)

  1. 一种肿瘤细胞来源的微颗粒，其包含凋亡的肿瘤细胞所释放的细胞囊泡，所述细胞囊泡在其表面表达刺突蛋白结合受体；
    优选地，所述刺突蛋白是肺炎病毒刺突蛋白；
    更优选地，所述肺炎病毒选自：SARS-CoV、SARS-CoV-2、或其变体；
    最优选地，所述刺突蛋白结合受体是血管紧张素转换酶2(ACE2)或其结合片段；
    所述肿瘤细胞是肺癌细胞或结肠癌细胞。
  2. 根据权利要求1所述的肿瘤细胞来源的微颗粒，其中：
    优选地，所述肺癌细胞选自：NCI-H196、NCI-H292、NCI-H460、NCI-H446、NCI-H1299、NCI-H1650、H1792、NCI-H3255、A427、A549、0225-02Sp、2F7、95-D、SPCA-1、LLC、Calu-1、Calu-3、L1022、PC9R、MSTO-211H、TKB-1；更优选，Calu-3、A549；
    优选地，所述结肠癌细胞选自：HCT116、HCT116/FU、HCT-8/FU、LoVo、LoVoADR、SW480、SW620、CaCo-2、RKO-E6、RKO-AS45-1、FET、HT55、HT115、HT-29、COLO 205、KM12、CL-40、KM12-SM、COLO320DM、NCI-H508、SW1417、COLO394、WiDr；更优选，Caco-2。
  3. 根据权利要求1所述的肿瘤细胞来源的微颗粒，所述细胞囊泡的平均粒径为200nm至800nm，优选300nm、400nm、500nm、600nm、700nm。
  4. 一种制备肿瘤细胞来源的微颗粒的方法，包括步骤：
    1)提供表达刺突蛋白结合受体的肿瘤细胞；
    2)使步骤1)的所述肿瘤细胞凋亡；
    优选地，通过选自以下任一项的方式使步骤1)的所述肿瘤细胞凋亡：紫外线照射、X射线照射、化疗药物；
    3)收集凋亡的肿瘤细胞所释放的细胞囊泡；优选地，通过离心收 集凋亡的肿瘤细胞所释放的细胞囊泡，所述细胞囊泡的平均粒径为200nm至800nm，优选300nm、400nm、500nm、600nm、700nm。
  5. 根据权利要求4所述的方法，其中：
    所述刺突蛋白是肺炎病毒刺突蛋白；
    所述肺炎病毒选自：SARS-CoV、SARS-CoV-2、或其变体；
    优选地，所述刺突蛋白结合受体是ACE2或其结合片段；
    所述肿瘤细胞选自：肺癌细胞、结肠癌细胞；
    所述肺癌细胞选自：NCI-H196、NCI-H292、NCI-H460、NCI-H446、NCI-H1299、NCI-H1650、H1792、NCI-H3255、A427、A549、0225-02Sp、2F7、95-D、SPCA-1、LLC、Calu-1、Calu-3、L1022、PC9R、MSTO-211H、TKB-1；更优选，Calu-3、A549；
    优选地，所述结肠癌细胞选自：HCT116、HCT116/FU、HCT-8/FU、LoVo、LoVo ADR、SW480、SW620、CaCo-2、RKO-E6、RKO-AS45-1、FET、HT55、HT115、HT-29、COLO 205、KM12、CL-40、KM12-SM、COLO320DM、NCI-H508、SW1417、COLO394、WiDr；更优选，Caco-2。
  6. 根据权利要求4所述的方法，其中，步骤3)中，通过以下方式收集细胞囊泡，包括步骤：
    在2至8℃，以800至1000g对步骤2)所得凋亡的肿瘤细胞离心5至15分钟，以去除完整细胞，得到第一产物；
    在2至8℃，以12000至15000g对第一产物离心1至3分钟，以去除细胞碎片，得到第二产物；
    在2至8℃，以12000至15000g对第二产物离心40至80分钟，以收集释放的细胞囊泡。
  7. 一种肿瘤细胞来源的微颗粒，其是通过权利要求4至6中任一项所述的方法制备所得。
  8. 权利要求1-3和7中任一项所述肿瘤细胞来源的微颗粒在制备药物中的用途，所述药物用于治疗病毒性肺炎；
    所述病毒选自：SARS-CoV、SARS-CoV-2、或其变体。
  9. 一种药物组合物，其包含：
    可药用载体；和
    权利要求1-3和7中任一项所述肿瘤细胞来源的微颗粒；
    所述药物组合物制备成肺施用的剂型。
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CN111875680A (zh) * 2020-08-08 2020-11-03 武汉圣润生物科技有限公司 一种预防新型冠状病毒微颗粒的制备方法及应用
CN112522203A (zh) * 2020-11-23 2021-03-19 中山大学附属第五医院 表达嵌合抗原受体的细胞囊泡及其制备方法和应用

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