Use of cellular microparticles in the treatment of respiratory viral pneumonia

Abstract

The use of cellular microparticles in the treatment of respiratory viral pneumonia has been disclosed. Tumor cell-derived microparticles (MPs) include cellular vesicles released by apoptotic tumor cells. The cellular vesicles express spike protein-binding receptors on their surfaces. MPs have high biosafety. In addition, it has been found that the receptors on the surface of microparticles have virus adsorption ability, and macrophages can efficiently take up MPs, which can be used to treat viral pneumonia.

Description

[Technical Field]

[0001] This application relates to the fields of biology, medicine, and clinical practice. Specifically, it relates to the use of cellular microparticles in the treatment of coronavirus disease 2019 infection. [Background technology]

[0002] Viral pneumonia can occur in large-scale epidemics, or as sporadic and endemic cases. While it can occur at any time of year, it is more common in winter and spring. Initially, viral pneumonia is primarily caused by a viral infection of the upper respiratory tract, and the virus spreads downwards towards the lungs, leading to pneumonia.

[0003] Viral pneumonia can be transmitted through droplets. Clinical signs are generally mild, mainly consisting of symptoms similar to those of respiratory illness, such as headache, fatigue, fever, and cough. Respiratory infections are one of the leading causes of death worldwide, and patients with severe pneumonia, in particular, have a high mortality rate and serious long-term consequences. Currently, viruses such as influenza virus and coronavirus, which are the main pathogens causing localized epidemics of severe pneumonia, are highly transmissible and have a high mortality rate.

[0004] Coronavirus disease 2019 (COVID-19) is an acute respiratory infection caused by a new coronavirus (SARS-CoV-2) that emerged in 2019.

[0005] SARS-CoV-2 is a novel coronavirus strain that has never been seen in humans before, and its main routes of transmission are respiratory droplets and contact. COVID-19 primarily presents as fever, dry cough, and fatigue, with a small number of patients also experiencing nasal congestion, runny nose, diarrhea, and other upper respiratory and gastrointestinal symptoms. Severely ill patients usually develop respiratory distress within about a week, and in severe cases, the disease rapidly progresses to acute respiratory distress syndrome, septic shock, coagulation disorders, multiple organ failure, etc., ultimately leading to death. SARS-CoV-2 is highly transmissible and has a high mortality rate.

[0006] Following the SARS-CoV-2 pneumonia pandemic, despite tremendous efforts in drug development across various countries, there is still no effective drug to treat SARS-CoV-2 infection. Significant progress has been made in vaccine research and development for the prevention and treatment of COVID-19, with China granting marketing approval for four COVID-19 vaccines, including three inactivated vaccines and one adenovirus vector vaccine. While vaccines can significantly prevent the spread of the virus, there is still a need to explore more effective treatment regimens for patients already infected. Currently, drug development for COVID-19 primarily involves screening small molecule compounds against viral replication and packaging, but many of these compounds face challenges due to uncertainty and safety concerns. While many drugs demonstrate good anticoronavirus activity in vitro, they often lack value in in vivo applications in humans. Some drugs are used in clinical antiviral treatments, but these are not specific to SARS-CoV-2, and their side effects are not negligible. [Overview of the project] [Problems that the invention aims to solve]

[0007] Therefore, it is necessary to explore unconventional treatment strategies for COVID-19 patients. [Means for solving the problem]

[0008] This invention provides tumor cell-derived microparticles, which express spike protein-binding receptors on their surface, act as targeting agents, and provide easier access to pneumonia treatment sites, derived from cellular vesicles of apoptotic tumor cells.

[0009] Those skilled in the art know that cells are composed of a cell membrane that encloses the cellular contents, that the cell membrane consists of protein molecules embedded in a phospholipid bilayer, and that the spherical structure of the cell is maintained by tractional forces formed by protein fiber filaments called the cytoskeleton. When a cell is stimulated by an external signal (e.g., chemotherapeutic drugs, ultraviolet light, radiation) and undergoes apoptosis, the protein fiber filaments are destroyed or lose their adhesion, the tractional forces disappear, and as a result, the local cell membrane structure swells outward, protrudes, and encloses the cellular contents in the form of vesicles. These vesicles are approximately 100 to 1000 nanometers in size and are referred to as "cellular vesicles" in this application.

[0010] The cellular vesicles described above are used as carriers to express targeting agents on their surface, further promoting the targeting of therapeutic targets. This type of vesicle cannot enter normal tissue (permeability is approximately 5 to 10 nm), does not cause damage to normal tissue, and therefore avoids the side effects caused by exogenous carriers such as nanomaterials.

[0011] In some embodiments, the cellular vesicles originate from tumor cells, and in particular, the tumor cells originate from the same type of tissue as the tissue at the site of treatment. In some embodiments, the cellular vesicles can readily come into contact with the cell membrane of the site of treatment in the patient's body. In some specific embodiments, the site of treatment in the patient is the lung. In some embodiments, the tumor cells are lung cancer cells. Therefore, the cellular vesicles originate from lung cancer cells.

[0012] In other embodiments, the type of tumor cells used to prepare the cellular vesicles may differ from the type of cells at the site where the cellular vesicles are administered. For example, the cellular vesicles of the present invention are prepared from colon cancer cells, but the cellular vesicles can be administered to the lungs.

[0013] In certain embodiments, tumor cells used to prepare cellular vesicles contain oxysterols, such as cholesterol with hydroxylation modifications. For example, tumor cells used to prepare cellular vesicles contain 25-hydroxylated cholesterol.

[0014] In the art, many well-known and available human tumor cell lines exist, and their oxysterol content varies. In some embodiments, tumor cell lines rich in oxysterols (e.g., hydroxylated cholesterol) can be selected as material for the preparation of cellular vesicles.

[0015] In some embodiments, the tumor cells are lung cancer cells, for example, 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, and TKB-1. In a particular embodiment, the tumor cells are oxysterol-rich Calu-3. In a particular embodiment, the tumor cells are oxysterol-rich A549.

[0016] In some embodiments, the tumor cells are colon cancer cells. The colon cancer cells are selected from the group consisting of 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, and WiDr. In a particular embodiment, the tumor cells are oxysterol-rich CaCo-2.

[0017] In some embodiments, the targeting agent is a spike protein-binding receptor expressed on the surface of a vesicle.

[0018] In some embodiments, the spike protein binding receptor expressed on the extracellular vesicles may be either naturally expressed or recombinantly expressed.

[0019] In some embodiments, the spike protein is the spike protein of the pneumonia virus (S, spike).

[0020] In some embodiments, the virus is selected from the group consisting of SARS-CoV, SARS-CoV-2, and variants thereof.

[0021] In some specific embodiments, the spike protein binding receptor is angiotensin-converting enzyme 2 or a binding fragment thereof.

[0022] In some specific embodiments, ACE2 is human ACE2. This term encompasses native human ACE2 or native variants thereof, as well as artificially expressed human ACE2 or variants thereof, such as recombinant human ACE2 or variants thereof expressed in vitro.

[0023] As used herein, the "binding fragment of angiotensin-converting enzyme 2" refers to a fragment of angiotensin-converting enzyme 2 as long as it still retains its ability to specifically bind to the spike protein. The binding fragment of angiotensin-converting enzyme 2 is also within the scope of the present application. Angiotensin-converting enzyme 2 or a binding fragment thereof in the present application may be naturally expressed, recombinantly expressed, or genetically engineered.

[0024] In some embodiments, the tumor cells are selected from lung cancer cells and colon cancer cells.

[0025] The present application provides a method for preparing microparticles derived from tumor cells by causing tumor cells to undergo apoptosis using any feasible means to obtain extracellular vesicles.

[0026] In some specific embodiments, a method for preparing microparticles derived from tumor cells, 1) A step to prepare tumor cells that express spike protein-binding receptors, 2) The step of inducing apoptosis in the tumor cells of step 1), 3) A process for collecting cellular vesicles released by apoptotic tumor cells. A method is provided that includes this.

[0027] In some specific embodiments, cells are induced by any well-known means in the art. Non-limiting examples include ultraviolet irradiation, X-ray irradiation, or chemotherapeutic agents such as dexamethasone. In the method of the present application, apoptosis can be induced without introducing exogenous substances into tumor cells, and therefore irradiation is preferred (e.g., ultraviolet irradiation, X-ray irradiation). In some specific embodiments, the duration and intensity of irradiation can be determined by those skilled in the art according to common practice.

[0028] In certain embodiments of the present application, ultraviolet light or chemotherapeutic agents are preferably used to induce apoptosis of tumor cells. For the recovery of cell vesicles, isolation may be performed by using an ultracentrifuge under low temperature conditions (or room temperature conditions). Preferably, the cell vesicles are recovered by centrifugation at low temperature conditions (e.g., about 4°C) with a centrifugal force of 100 to 100,000 g.

[0029] In some specific embodiments, cellular vesicles released by apoptotic tumor cells are recovered by centrifugation, and the average particle size of the cellular vesicles is 200 nm to 800 nm, preferably 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, and 800 nm ± 10%.

[0030] In some specific embodiments, a method for preparing microparticles derived from tumor cells, 1) A step of preparing human tumor cells, which are lung cancer cells or colon cancer cells, that express human ACE2 or its binding fragment. 2) The step of inducing apoptosis in the tumor cells of step 1), 3) Cellular vesicles released by apoptotic tumor cells, Step 2) The apoptotic tumor cells obtained in step 2) are centrifuged at 2 to 8°C at 800 to 1000 g for 5 to 15 minutes to remove intact cells and obtain the first product. The first product is centrifuged at 12,000 to 15,000 g for 1 to 3 minutes at 2 to 8°C to remove cell debris and obtain the second product. The second product is centrifuged at 2 to 8°C at 12,000 to 15,000 g for 40 to 80 minutes to collect the released cellular vesicles. A process of recovery by means including A method is provided that includes this.

[0031] In some specific embodiments, cellular vesicles released by apoptotic tumor cells The obtained apoptotic tumor cells are centrifuged at 2 to 8°C at 1000g for 10 minutes to remove intact cells and obtain the first product. The first product is centrifuged at 14,000 g for 2 minutes at 2 to 8°C to remove cell debris and obtain the second product. The second product is centrifuged at 14,000 g for 60 minutes at 2 to 8°C to collect the released cellular vesicles. The average particle size of the cell vesicles recovered by means including the above steps falls in the range of 100 nm to 1000 nm, particularly in the range of 200 nm to 800 nm.

[0032] This invention provides microparticles derived from tumor cells prepared by the method described above.

[0033] This application provides a pharmaceutical composition containing microparticles derived from tumor cells in accordance with this application.

[0034] According to a preferred embodiment of the pharmaceutical composition of the present application, the pharmaceutical composition comprises cellular vesicles or microparticles derived from tumor cells.

[0035] The recovered fine particles can be prepared into pharmaceutical compositions, particularly spray formulations, according to conventional means.

[0036] In a preferred embodiment of the present application, the average particle size in a pharmaceutical composition formed by microparticles derived from tumor cells is 100 to 1000 nanometers, and non-limiting examples that may be mentioned are 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 900, 950, and 1000 ± 10% nm.

[0037] The drug composition provided herein may be administered according to conventional clinical treatment methods, for example, in pneumonia, it may be administered directly by nasal drops, spray, or perfusion into the lungs. The dose used for administration may be determined by a healthcare professional.

[0038] In certain embodiments, the microparticles, cell vesicles, or pharmaceutical compositions provided herein are prepared in a dosage form suitable for pulmonary administration.

[0039] For example, inhalable powders for pulmonary administration are produced by conventional techniques such as jet milling, spray drying, solvent precipitation, and supercritical fluid concentration. Metered-dose inhalers made using dry powder inhalers (DPIs), such as those based on Nektar®, Vectura® (Gyrohaler®), and GSK® (Discus®), or Astra® (Turbohaler®), contain the microparticles, cellular vesicles, or pharmaceutical composition of the present application in a suitable carrier (e.g., mannitol, sucrose, or lactose), which are delivered to the surface of the terminal alveoli. Ultrasonic nebulizers can also be used to deliver liposome-containing (or non-liposome-containing) solution preparations to the lungs.

[0040] According to certain embodiments of the present invention, microparticles derived from A549 cells are provided that can be used as a therapeutic means for patients with COVID-19 infection. Angiotensin-converting enzyme 2 (ACE2), present on the surface of the microparticles, is a receptor that can bind to the surface protein of SARS-CoV-2 and mediate viral entry. Thus, the MPs can adsorb the SARS-CoV-2 virus and limit the spread of the virus in the body. The virus-carrying microparticles are efficiently taken up by macrophages, delivered to lysosomes, and degraded, thereby enabling effective treatment of COVID-19.

[0041] In this application, the term "microparticles (MPs)" refers to vesicle-like structures with an average particle size of 100 to 1000 nm that separate from the membrane surface of eukaryotic cells during activation or apoptosis. Microparticles are considered to be carriers of bioinformation, mediating the transmission and exchange of bioinformatics between different types of cells. Due to their characteristics such as high biocompatibility, low immunogenicity, and targeting ability, MPs are used as drug carriers, and microparticles derived from tumor cells loaded with antitumor drugs have good antitumor effects and are applied in clinical practice.

[0042] In some embodiments, the microparticle formulation showed 5 × 10 in animal experiments. 6 The dose is 50 μl, administered once daily for 5 days. Those skilled in the art can determine the unit dose for human subjects based on animal experiments. [Brief explanation of the drawing]

[0043] [Figure 1] This figure shows ACE2 expression in microparticles (MPs) derived from A549 cells, with Western blot analysis indicating ACE2 expression in the MPs. [Figure 2A]This figure shows that SARS-CoV-2 can be adsorbed by MP. After incubating AMP with SARS-CoV-2 and filtering the solution through a 0.1 Mp type filter, real-time PCR analysis shows that viral RNA can be detected with incubated MP but not with unincubated MP. [Figure 2B] This figure shows that SARS-CoV-2 can be adsorbed by MP. Immunofluorescence staining further supports the binding of the viral S protein to ACE2 on MP. [Figure 3] This figure shows that MPs adsorb virions and deliver them to alveolar macrophages. In vitro incubation of alveolar macrophages with virus-adsorbing MPs shows that macrophages can take up virus-containing MPs within 10 minutes, but isolated primary type II alveolar epithelial cells have difficulty taking up MPs. [Figure 4] This figure shows that SARS-CoV-2 virus adsorbed by MP is inactivated in alveolar macrophages. After incubating SARS-CoV-2 and SARS-CoV-2 / MP with alveolar macrophages for 0.5 hours, 1 hour, and 4 hours, SARS-CoV-2 was able to replicate in the alveolar macrophages, while the amount of SARS-CoV-2 bound to MP in the alveolar macrophages was reduced. Probe 1 targets the sense sequence of the virus to evaluate viral distribution (green), and probe 2 targets the antisense sequence of the virus to show viral replication (red) (those skilled in the art can design probes according to conventional design principles). [Figure 5A] This figure shows a confocal microscope image with a 5 μm scale bar. [Figure 5B]This figure shows that cholesterol-25-hydroxylase in irradiated A549-ACE2-OE cells is significantly upregulated, and the 25-hydroxycholesterol content of the extracted microparticles is significantly increased. Note: 293T is the human renal epithelial 293 cell line (a cell line with low hydroxylated cholesterol content), 293T-UV is 293T cells after 1 hour of UV irradiation, A549-OE is cells that overexpress A549 ACE2, A549-OE-UV is cells that overexpress A549 ACE2 after 1 hour of UV irradiation, 293-MP is microparticles derived from 293T cells, and AO-MP is microparticles derived from cells that overexpress A549 ACE2. [Figure 5C] This figure shows that cholesterol-25-hydroxylase in irradiated A549-ACE2-OE cells is significantly upregulated, and the 25-hydroxycholesterol content of the extracted microparticles is significantly increased. Note: 293T is the human renal epithelial 293 cell line (a cell line with low hydroxylated cholesterol content), 293T-UV is 293T cells after 1 hour of UV irradiation, A549-OE is cells that overexpress A549 ACE2, A549-OE-UV is cells that overexpress A549 ACE2 after 1 hour of UV irradiation, 293-MP is microparticles derived from 293T cells, and AO-MP is microparticles derived from cells that overexpress A549 ACE2. [Figure 5D]This figure shows that cholesterol-25-hydroxylase in irradiated A549-ACE2-OE cells is significantly upregulated, and the 25-hydroxycholesterol content of the extracted microparticles is significantly increased. Note: 293T is the human renal epithelial 293 cell line (a cell line with low hydroxylated cholesterol content), 293T-UV is 293T cells after 1 hour of UV irradiation, A549-OE is cells that overexpress A549 ACE2, A549-OE-UV is cells that overexpress A549 ACE2 after 1 hour of UV irradiation, 293-MP is microparticles derived from 293T cells, and AO-MP is microparticles derived from cells that overexpress A549 ACE2. [Figure 5E] This figure shows that knocking out cholesterol-25-hydroxylase (abbreviated as CH25H) eliminates the effect of microparticles that upregulate the pH of endosomes. Ctrl is the group with PBS only, SGCtrl is the group with control microparticles derived from A549, SG1 is the group with microparticles derived from A549-CH25H knockout cells, and SG2 is the group with microparticles derived from A549-CH25H knockout cells. [Figure 6A] This figure shows that MPs enhance the degradation of the SARS-CoV-2 virus by lowering the pH within lysosomes. Treatment of alveolar macrophages by MPs lowers the pH of lysosomes. [Figure 6B] This figure shows that MP enhances the degradation of the SARS-CoV-2 virus by lowering the pH within lysosomes. Lysosomes isolated from macrophages treated with MP inactivate the virus more efficiently than lysosomes isolated from control macrophages. [Figure 7A]This figure shows the in vivo efficacy of MP in treating SARS-CoV-2 virus infection. Five days after SARS-CoV-2 infection, MP was injected into the nasal cavity of mice for five consecutive days. H&E staining shows a reduction in peribronchial and perivascular inflammatory cell infiltration, as well as a reduction in lung histopathological damage, in the treated mice. Ctrl represents the control group, and Mock represents the PBS-treated group. [Figure 7B] This figure shows the in vivo efficacy of MP in treating SARS-CoV-2 virus infection. Five days after SARS-CoV-2 infection, MP was injected into the nasal cavity of mice for five consecutive days. Consistent with morphological improvement, RNA Scope showed a reduction in viral load in the lungs. Ctrl represents the control group, and Mock represents the PBS-treated group. [Figure 8A] This figure shows the levels of TNF-α, IL-1β, and IL-6 inflammatory factors in in vitro macrophages that have taken up SARS-CoV-2-carrying polyposis (MPs) compared to macrophages that have taken up SARS-CoV-2 alone. [Figure 8B] This figure shows the levels of TNF-α, IL-1β, and IL-6 inflammatory factors in lung tissue from mice treated with MP. Note: Ctrl is the control group, and Mock is the PBS-treated group. [Modes for carrying out the invention]

[0044] Various cell lines, agents, and experimental animals used in the examples We purchased the mouse macrophage cell line Raw264.7 and the A549 human lung adenocarcinoma cell line from the China Center for Type Culture Collection (CCTCC).

[0045] Female ICR, hACE2 transgenic mice, 6 to 8 weeks old, were purchased from the Medical Laboratory Animal Center of the Chinese Academy of Medical Sciences (Beijing). The study of these virus-free mice was approved by the Animal Protection and Utilization Committee of the Chinese Academy of Medical Sciences. [Examples]

[0046] ACE2 is expressed in microparticles (MPs) derived from A549 cells. 1. Experimental Procedure The coding sequence for human ACE2 was amplified and inserted into the plasmid pLV-EF1α-IRES-Puro. This plasmid was then transiently expressed in 293T cells to obtain a virus containing the human ACE2 gene. The lentivirus containing human ACE2 was transduced into A549 cells, which were then screened with 1 μg / ml puromycin to obtain cell clones with high ACE2 expression, i.e., A549-ACE2-OE.

[0047] To construct a stable knockout ACE2 cell line, knockout RNAs were designed for well-known human ACE2 gene sequences in a database. The RNAs were cloned into a pSpCas9(BB)-2A-GFP vector plasmid, which was used to transform cells. After 48 hours, GFP-positive cells were sorted by flow cytometry using BD Biosciences' FACSAria III. Candidate knockout cells were confirmed by Western blotting or immunofluorescence to obtain A549-ACE2-SG (ACE2 knockout A549 cells).

[0048] A549, A549-ACE2-OE, and A549-ACE2-SG each have a rated load of 300 J / m². 2The cells were irradiated with ultraviolet light for 1.5 hours, and the supernatant was collected after 18 hours. The supernatant was centrifuged at 1000×g for 10 minutes to remove cells, and then centrifuged at 14000×g for 2 minutes to remove residue. Next, the supernatant was centrifuged at 14000×g for 60 minutes at 4°C to prepare each type of MP. The MPs were washed three times and suspended in culture medium for subsequent experiments.

[0049] A549 cells and each type of MP were dissolved in lysis buffer and sonicated. Protein concentrations were determined using a BCA kit. The proteins were then degraded on an SDS-PAGE gel and transferred to a nitrocellulose membrane. The nitrocellulose membrane was blocked with 5% bovine serum albumin and detected overnight with antibody to detect ACE2 expression in MP, with cells used as a positive control.

[0050] 2. Experimental Results Western blot analysis revealed ACE2 expression in MP and the presence of ACE2 in A549-derived microparticles obtained using this method (Figure 1). [Examples]

[0051] SARS-CoV-2 virus can be adsorbed by MPs. 1. Experimental Procedure MP obtained from A549-ACE2-OE was incubated with SARS-CoV-2 virus at 37°C for 30 minutes, and then filtered through a 0.1 μm filter membrane. The filter selectively allowed viral particles to pass through but prevented the MP from passing through. SARS-CoV-2 virus that was not incubated with MP was used as a control group and was also filtered through the filter membrane. The viral load on the membranes of the different groups was detected by real-time quantitative PCR.

[0052] MP(5×10 5The cells were incubated with recombinant SARS-CoV-2 spike protein (0.1 μg), fixed, and then stained with anti-ACE2 protein antibody (red) and anti-spike protein antibody (green). Fluorescence was determined by super-resolution structured illumination microscopy; the scale bar is 2 μm.

[0053] 2. Experimental Results After incubation of AMP with SARS-CoV-2, Q-PCR analysis showed that viral RNA was detected in incubated MPs but not in unincubated MPs (Figure 2A). Immunofluorescence staining confirmed the binding of viral S protein to ACE2 on MPs (Figure 2B). [Examples]

[0054] MPs adsorb virions and deliver them to alveolar macrophages. 1. Experimental procedure: Primary alveolar macrophages were isolated from mouse bronchoalveolar lavage fluid. Mice were anesthetized, immediately lavaged, and the trachea was transected. The lungs were washed five times with 1 ml of PBS, and the lavage fluid was centrifuged at 600 × g for 5 minutes at 4°C. The recovered cells were suspended in RPMI1640 complete medium and cultured in culture plates for 2 hours, then gently washed with PBS to remove non-adherent cells.

[0055] Primary alveolar epithelial cells were isolated from hACE2 mice perfused with 10 ml of cold PBS through the right ventricle. Lung tissue was filled with 2 ml of racemase and a low gelling temperature agarose gel, then incubated with 2 ml of racemase at 37°C for 20 minutes. The lung tissue was then pulverized, and the homogenate was filtered through 70 and 40 μm nylon mesh. Leukocytes, monocytes / macrophages, NK cells, neutrophils, endothelial cells, and erythrocytes were removed from the cell suspension using biotin-labeled antibodies and magnetic beads.

[0056] Primary alveolar macrophages and primary alveolar epithelial cells were isolated from ICR mice. After treatment with PKH67-labeled A-OE-MP for 10 minutes, 30 minutes, and 2 hours, images were captured under a confocal microscope; the scale bar is 5 μm.

[0057] 2. Experimental results: In vitro incubation of alveolar macrophages with virus-adsorbing MPs showed that macrophages could take up virus-containing MPs within 10 minutes, while isolated primary type II alveolar epithelial cells had difficulty taking up MPs even after 2 hours (Figure 3). [Examples]

[0058] SARS-CoV-2 virus adsorbed by MPs is inactivated in alveolar macrophages. 1. Experimental procedure: MP(5×10 5 ) SARS-CoV-2 (5 × 10 4 TCID 50 The cells were incubated with SARS-CoV-2 at 37°C for 30 minutes, then alveolar macrophages were added and treated for 30 minutes, 1 hour, and 4 hours. MPs not incubated with SARS-CoV-2 were used as a control group. Cells were fixed with 4% paraformaldehyde and incubated with hydrogen peroxide at room temperature for 10 minutes, and RNA was analyzed in situ using the RNAScope kit. Probe 1 targeted the sense sequence of the virus to assess viral distribution (green), and probe 2 targeted the antisense sequence of the virus to show viral replication (red), with the scale bar being 5 μm (those skilled in the art can design probes 1 and 2 according to conventional design principles).

[0059] 2. Experimental results: After incubating SARS-CoV-2 and SARS-CoV-2 / MP with alveolar macrophages for 0.5 hours, 1 hour, and 4 hours, SARS-CoV-2 alone could replicate in alveolar macrophages, while the amount of SARS-CoV-2 bound to MP was reduced in alveolar macrophages (Figure 4). [Examples]

[0060] MPs prevent virus evasion by increasing the pH of endosomes. Macrophages were pre-treated with microparticles for 30 minutes, then labeled with pHrodo® Red dextran for 10 minutes, and imaged using a confocal microscope. The scale bar is 5 μm.

[0061] A549-ACE2-OE cells are plated in a 6-well plate (5 x 10 5 ) Sow seeds and irrigate at 300 J / m 2 The cells were irradiated with ultraviolet light for 1 hour. RNA was collected after 18 hours, and the CH25H content was detected by qPCR. Simultaneously, microparticles were extracted using this method, and the 25-hydroxycholesterol (25HC) content in the MP was detected by mass spectrometry. CH25H in A549 cells was knocked out using the CRISPR-Cas9 system, and then the MP was extracted and used to treat macrophages for 30 minutes. Subsequently, pHrodo® Red dextran was added for labeling, and imaging was performed using a confocal microscope (Figure 5A).

[0062] The results showed that microparticles can inhibit viral replication by increasing the pH of endosomes, preventing the virus from escaping into the cytoplasm. After treatment with microparticles, the pH of macrophage endosomes increased. Oxysterols carried by the microparticles affected the pH of endosomes. Figures 5B, 5C, and 5D show that cholesterol-25-hydroxylase was significantly upregulated in irradiated A549-ACE2-OE cells, and the content of 25-hydroxycholesterol in the extracted microparticles was also significantly increased. Figure 5E shows that the effect of microparticles in upregulating the pH of endosomes disappeared by knocking out CH25H. [Examples]

[0063] MP enhances the degradation of the SARS-CoV-2 virus by lowering the pH within lysosomes. 1. Experimental Procedure Alveolar macrophages were pretreated with MP obtained from A549-ACE2-OE for 30 minutes, and alveolar macrophages not treated with MP were used as a control group. Macrophages were stained with LysoSensor® Yellow / Blue DND-160, and the pH value was detected using a microplate reader. The pH of macrophage lysosomes was determined using LysoSensor® Yellow / Blue DND-160 with a lysosomal pH meter. The results showed that the pH of living cells was dependent on two-photon excitation spectroscopy.

[0064] Lysosomes were isolated and purified, adherent cells were digested with trypsin, and then washed with ice-cold PBS. The cell pellet was resuspended and placed in a homogenizer for disruption. The cell homogenate was centrifuged at 1000×g for 10 minutes, and the supernatant was centrifuged at 20000×g for 20 minutes to prepare lysosome particles and other organelles. A density gradient was established and centrifuged at 150000×g for 4 hours in a SW50.1 rotor to remove the top (lowest density) band, which was diluted in PBS. After washing, the lysosomes were centrifuged at 20000×g for 20 minutes for isolation and purification.

[0065] Lysosomes isolated from macrophages treated with MP and lysosomes isolated from control macrophages were each incubated with SARS-CoV-2 at 37 °C for 30 minutes, and then virus E6 was added and infected for 48 hours. The cells were stained with anti-NP antibody. The scale bar was 5 μm.

[0066] 2. Experimental results Treatment of alveolar macrophages with MP decreased the pH of lysosomes (Figure 6A), and lysosomes isolated from macrophages treated with MP had a lower amount of virus than lysosomes isolated from control macrophages (Figure 6B).

Example

[0067] In vivo efficacy of MP in the treatment of SARS-CoV-2 virus infection 1. Experimental procedure hACE2 mice were infected intratracheally with SARS-CoV-2 (1×10 5 TCID 50 ), and then treated with a control group (PBS) or MP (5×10 6 ). The treatment was performed once a day for 5 consecutive days (n = 5 animals per group). After 5 days of treatment, the mice were sacrificed, and the lung tissues were fixed and subjected to HE staining.

[0068] After fixing the lung tissue, in situ RNA analysis was performed using the RNAScope kit. Probe 1 targeted the viral sense sequence to assess viral distribution (green), and probe 2 targeted the viral antisense sequence to show viral replication (red). The scale bar is 5 μm.

[0069] 2. Experimental Results H&E staining showed reduced peribronchial and perivascular inflammatory cell infiltration and mitigated pathological damage to lung tissue in treated mice (Figure 7A). Consistent with morphological improvements, RNAScope showed a reduction in viral load in the lungs (Figure 7B). [Examples]

[0070] Macrophages do not produce an inflammatory response when they eliminate SARS-CoV-2 that has been adsorbed to MPs. SARS-CoV-2 (1 × 10⁻¹⁰ 5 TCID 50 ) macrophages (1 x 10 5 The RNA was added to the solution for 24 hours. RNA was extracted by TRIZOL, and the levels of TNF-α, IL-1β, and IL-6 were detected by qPCR.

[0071] SARS-CoV-2 (1x10) in hACE2 mice 5 TCID 50 ) were infected in the trachea, and then the control group (PBS) or MP (5 × 10 6 Each animal was treated with [specific method]. The treatment was performed once a day for 5 consecutive days (n = 5 animals per group). The mice were sacrificed 5 days after treatment. RNA from lung tissue was extracted by TRIZOL, and the levels of TNF-α, IL-1β, and IL-6 were detected by qPCR.

[0072] Figure 8A shows that levels of TNF-α, IL-1β, and IL-6 inflammatory factors were significantly reduced in in vitro macrophages that had taken up MP carrying SARS-CoV-2 compared to macrophages that had taken up SARS-CoV-2 alone. Figure 8B shows that levels of TNF-α, IL-1β, and IL-6 inflammatory factors were significantly downregulated in lung tissue from mice treated with MP.

[0073] In summary, the technical solution of this application has the following effects:

[0074] 1. The A549 cell-derived microparticles (MPs) provided herein are used to treat SARS-CoV-2 infection as a novel and unprecedented treatment strategy, by utilizing their properties of adsorbing the SARS-CoV-2 virus and enhancing the ability of alveolar macrophages to eliminate SARS-CoV-2.

[0075] 2. After viral infection, intratracheal infusion of MP allows ACE2 present on the surface of microparticles to bind to the SARS-CoV-2 surface S protein, adsorbing the viral particles and thereby limiting further viral spread within the body. The virus-carrying microparticles are efficiently taken up by macrophages and delivered to lysosomes. MP enhances the ability of macrophages to eliminate SARS-CoV-2 by regulating the pH value of lysosomes, thereby achieving clinical treatment of COVID-19. In this way, MP can effectively reduce the mortality rate of COVID-19 patients and has a promising prospect for clinical application.

[0076] 3. The microparticles of this invention are derived from cells and possess characteristics such as high biocompatibility, low immunogenicity, and targeting ability, resulting in high safety, toxicity, and no side effects.

Claims

1. Tumor cell-derived microparticles containing cellular vesicles released by apoptotic tumor cells, wherein the cellular vesicles express spike protein-binding receptors on their surface. The spike protein-binding receptor is angiotensin-converting enzyme 2 (ACE2) or its binding fragment. The tumor cells are lung cancer cells or colon cancer cells. Apoptotic tumor cells are tumor cells that have been irradiated with ultraviolet light. Microparticles derived from tumor cells.

2. Lung cancer cells are selected from the group consisting of 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, and TKB-1, Colon cancer cells are selected from the group consisting of 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, and WiDr. The tumor cell-derived microparticles according to claim 1.

3. The tumor cell-derived microparticles according to claim 1, wherein the average particle size of the cell vesicles is 200 nm to 800 nm.

4. 1) A step to prepare tumor cells that express spike protein-binding receptors, 2) A step of inducing apoptosis in the tumor cells of step 1), wherein the tumor cells of step 1) are inducing apoptosis by ultraviolet irradiation, 3) A process for collecting cellular vesicles released by apoptotic tumor cells. A method for preparing microparticles derived from tumor cells, including [a specific component].

5. The spike protein-binding receptor is ACE2 or a binding fragment thereof, The tumor cells are selected from a group consisting of lung cancer cells and colon cancer cells. The method according to claim 4.

6. Lung cancer cells were selected from the group consisting of 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, and TKB-1. Colon cancer cells are selected from the group consisting of 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, and WiDr. The method according to claim 5.

7. In step 3), the cell vesicles, Step 2) The apoptotic tumor cells obtained in step 2) are centrifuged at 2 to 8°C at 800 to 1000 g for 5 to 15 minutes to remove intact cells and obtain the first product. The first product is centrifuged at 12,000 to 15,000 g for 1 to 3 minutes at 2 to 8°C to remove cell debris and obtain a second product, and The second product is centrifuged at 2 to 8°C at 12,000 to 15,000 g for 40 to 80 minutes to collect the released cellular vesicles. The method according to claim 4, wherein the recovery is carried out by means including the method.

8. Use of tumor cell-derived microparticles according to any one of claims 1 to 3 in the preparation of a pharmaceutical for treating viral pneumonia, wherein the virus is selected from the group consisting of SARS-CoV, SARS-CoV-2, and variants thereof.

9. A pharmaceutically acceptable carrier, and Tumor cell-derived microparticles according to any one of claims 1 to 3 A pharmaceutical composition for pulmonary administration, including the above.

10. The pharmaceutical composition according to claim 9 for treating viral pneumonia, wherein the virus is selected from the group consisting of SARS-CoV, SARS-CoV-2, and variants thereof.

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