Lysosome-based antiviral formulations, methods of making and uses thereof

Abstract

The present application discloses a lysosome-based antiviral preparation, a preparation method and use thereof. The lysosome preparation disclosed by the present application first captures viruses by using the affinity of the virus-binding molecules modified on the surface of lysosomes to the ligand proteins mediating the entry of viruses into cells, then internalizes the viruses, completely isolates the viruses from host cells, and finally degrades the viruses by using the hydrolytic enzymes in lysosomes, thereby reducing the possibility of escape or persistent infection. In addition, the lysosome preparation of the present application will not lose efficacy due to virus mutation, and any virus variant combined with the virus-binding molecules is sensitive to the clearance mediated by the lysosome "trap", which can make up for the deficiency of current virus treatment and efficiently clear viruses.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical formulation technology, specifically to a lysosome-based antiviral formulation, a method for preparing the antiviral formulation, and its application in antiviral therapy, particularly in the treatment of coronavirus and influenza viruses. Background Technology

[0002] Viruses are among the major pathogens threatening human health. Vaccination is the primary measure to prevent viral infections such as coronaviruses. However, due to the rapid mutation of viruses like coronaviruses, antigenic drift can occur, posing a risk of vaccine ineffectiveness. Furthermore, vaccination is not suitable for the elderly, children, and other immunocompromised individuals, as well as severely ill patients already infected with the virus; therefore, more effective antiviral treatment strategies are urgently needed.

[0003] Currently, there is a need to develop agents that are not affected by viral mutations and can specifically and directly eliminate the virus, thereby inhibiting viral infection, alleviating viral symptoms, and improving the quality of life of patients.

[0004] The information in the background section is merely intended to illustrate the general background of the invention and should not be construed as an admission or implication in any way that such information constitutes prior art known to those skilled in the art. Summary of the Invention

[0005] To address at least some of the technical problems in the prior art, the present invention provides a lysosome-based antiviral preparation. Specifically, the present invention includes the following.

[0006] A first aspect of the present invention provides a lysosome-based antiviral formulation comprising a lysosome and a virus-binding molecule located on the surface of the lysosome or a precursor for generating the virus-binding molecule.

[0007] In some embodiments, in the lysosome-based antiviral formulation according to the present invention, the lysosomes are derived from any cell capable of producing lysosomes. In some embodiments, in the lysosome-based antiviral formulation according to the present invention, the cell is a macrophage, including at least one of primary macrophages, M1 macrophages, and engineered macrophages.

[0008] In some embodiments, in the lysosome-based antiviral formulation according to the present invention, the virus-binding molecule includes a receptor for a viral surface protein.

[0009] In some embodiments, in the lysosome-based antiviral formulation according to the present invention, the receptor binds to the lysosome via a linker molecule.

[0010] In some embodiments, in the lysosome-based antiviral formulation according to the present invention, the linker molecule includes a hydrophobic end, a spacer arm, and a binding domain, wherein the binding domain is capable of binding to the receptor.

[0011] In some embodiments, in the lysosome-based antiviral formulation according to the present invention, the receptor is a modified receptor.

[0012] In some embodiments, the lysosome-based antiviral formulation according to the present invention includes, wherein the precursor comprises sialyl cytidine monophosphate and optionally sialyl transferase.

[0013] A second aspect of the present invention provides a method for preparing the lysosome-based antiviral agent described in the first aspect.

[0014] A third aspect of the invention provides the use of lysosomes in the preparation of medicaments for the prevention and control of viral infections, wherein the lysosomes are modified to have virus-binding molecules on their surface.

[0015] A fourth aspect of the invention provides the use of a combination of a lysosome and a precursor for generating a virus-binding molecule on the surface of the lysosome in the preparation of a medicament for preventing and controlling viral infections.

[0016] In some embodiments, according to the intended use of the invention, the prevention and control includes: To reduce or eliminate the viral load in the subject's body; or Reduce or weaken viral activity in the subject.

[0017] This invention utilizes the affinity of virus-binding molecules for viral ligand proteins such as S / ACE2 to capture viruses, subsequently internalizing them to completely isolate them from host cells, and finally degrading the virus using lysosomal hydrolases, reducing the possibility of escape or persistent infection. Furthermore, the lysosomal formulation of this invention does not lose its efficacy due to viral mutations; any viral variant binding to the virus-binding molecules is sensitive to lysosomal "traps"-mediated clearance. In some embodiments, this invention addresses the shortcomings of current clinical anti-coronavirus treatments, efficiently clearing the virus. Attached Figure Description

[0018] Figure 1 Fluorescence confocal images of lysosomal preparations LAMP-2 and ACE2 prepared by chemical modification.

[0019] Figure 2 Fluorescence confocal images of LAMP-2 and ACE2, lysosomal preparations prepared by genetic engineering.

[0020] Figure 3 TEM images of lysosomes and lysosomal preparations.

[0021] Figure 4 Particle size and potential of lysosomes and lysosomal formulations (size at the top, zeta potential at the bottom).

[0022] Figure 5 Changes in the enzyme activities of ACE2 (left), proteases, and nucleases in lysosomal preparations before and after ACE2 protein modification.

[0023] Figure 6 The binding ability of lysosomal preparations to pseudoviruses was investigated.

[0024] Figure 7 TEM images and corresponding electron micrographs of pseudoviruses internalized in lysosomal preparations.

[0025] Figure 8 Pseudovirus protein levels.

[0026] Figure 9 Pseudovirus nucleic acid levels.

[0027] Figure 10 Confocal microscope image of virus-infected cells.

[0028] Figure 11 Flow cytometry results of virus-infected cells.

[0029] Figure 12 Imaging results of small animals after administration of lysosomal preparations.

[0030] Figure 13 Results of major organ distribution 9 hours after administration of lysosomal preparation.

[0031] Figure 14 Blood routine analysis after administration of lysosomal preparations (from left to right in the bar chart of each group, PBS, Lysosome, ACE2, LysoTRAP are represented).

[0032] Figure 15 Comprehensive biochemical analysis after administration of lysosomal preparations (from left to right in the bar charts of each group: PBS, Lysosome, ACE2, LysoTRAP).

[0033] Figure 16 HE section images of major tissues and organs after administration of lysosomal preparations.

[0034] Figure 17-18 The study demonstrated that the lysosomal formulation significantly inhibited viral infection at the animal level.

[0035] Figure 19The viral copy numbers of the lysosomal preparation groups are shown (from left to right: PBS, EIDD-2801, RBDantibody, h-lysoTRAP).

[0036] Figure 20 The lysosomal preparation group significantly inhibited viral S protein expression.

[0037] Figure 21 Nucleic acid load analysis of viral infection (from left to right: PBS, EIDD-2801, RBD antibody, h-lysoTRAP).

[0038] Figure 22 Immunofluorescence section analysis of viral infections.

[0039] Figure 23 Cluster analysis of the efficacy of each treatment group.

[0040] Figure 24 Confocal micrographs of LAMP-2 and SA, anti-influenza virus lysosomal preparations prepared by lysosomes and chemical modification.

[0041] Figure 25 Fluorescence confocal images of LAMP-2 and SA, anti-influenza virus lysosomal preparations prepared by lysosomes and genetic engineering.

[0042] Figure 26 Changes in body weight in mice after viral infection (from top to bottom: NC, LysoTRAP (SA), PBS).

[0043] Figure 27 Changes in lung weight and lung index in mice after viral infection.

[0044] Figure 28 Analysis of viral nucleic acid load in mice after viral infection. Detailed Implementation

[0045] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0046] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0047] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0048] Lysosome-based antiviral agents In a first aspect, the present invention provides a lysosome-based antiviral formulation, which is an antiviral formulation developed or prepared based on lysosomes, and is sometimes simply referred to herein as a "lysosome formulation". Furthermore, the lysosome formulation of the present invention can capture or further kill viruses; therefore, the lysosome-based antiviral formulation or lysosome formulation may also be referred to as a lysosome "trap". These three terms are used interchangeably herein.

[0049] The lysosomal formulation of the present invention comprises lysosomes and a virus-binding molecule located on the surface of the lysosomes or a precursor for generating the virus-binding molecule.

[0050] Virus binding molecules In this invention, the virus-binding molecules are generally located or immobilized on the surface of lysosomes through artificial means, or the surface of lysosomes is modified to have virus-binding molecules bound to it, or the amount of virus-binding molecules is increased to a level sufficient to effectively adsorb or capture viruses. The virus-binding molecules are not particularly limited; they can be general-purpose binding molecules targeting a variety of different viruses, or specific binding molecules targeting a particular virus.

[0051] In this invention, since the virus binding molecule is not particularly limited and can be any known type, the lysosomal preparation of this invention can be prepared as a preparation for various different viruses as needed. Therefore, the lysosomal preparation can be understood as a lysosomal preparation platform, and should not be understood as a specific preparation for a particular virus.

[0052] In some embodiments, the virus-binding molecule of the present invention is a viral protein receptor, such as a binding receptor for a protein on the surface of a virus, examples of which include, but are not limited to, angiotensin-converting enzyme 2 (ACE2), sialic acid, cholesterol transporter NPC1, CD4 protein, etc. Preferably, the viral receptor is a modified receptor capable of binding to or immobilizing on the surface of a lysosome. Exemplarily, the receptor is modified and covalently bound to a linker molecule. The linker molecule is designed to insert into the lysosomal membrane to bind to or immobilize it. In some embodiments, the linker molecule is designed to include a hydrophobic end, a spacer arm, and a binding domain. The hydrophobic end is located at the end of the linker molecule for insertion into the lysosomal membrane to immobilize the linker molecule. The spacer arm is generally located in the middle of the linker molecule and is not particularly limited as long as it has a spacer function; it can be, for example, a hydrophilic molecular chain. The binding domain is located at the other end of the linker molecule for binding to the viral receptor molecule.

[0053] In this invention, the lysosomal formulation can also be designed as a composition of multiple components, wherein at least a portion of the components are precursors for generating virus-binding molecules. Examples of such precursors include reagents or molecules for generating sialic acid on the lysosomal surface, including sialyl cytidine monophosphate and sialyl transferase. When both sialyl cytidine monophosphate and sialyl transferase are included, the amounts of both are not particularly limited; for example, the amounts of sialyl cytidine monophosphate and sialyl transferase are based on a weight ratio of 1-20:1-5. Generally, it is preferred that the amount of sialyl cytidine monophosphate is greater than that of sialyl transferase. For example, 1.5-20:1, 2-20:1, 5-20:1, etc.

[0054] In some embodiments, the lysosomal formulation of the present invention comprises lysosomes, sialyl cytidine monophosphate, and sialyl transferase. The mass ratio of lysosomes, sialyl cytidine monophosphate, and sialyl transferase is generally 50-1000:1-20:1-5. For example, 50-800:1-20:1-5, or even 50-500:1-20:1-5, or 100-800:6-15:1-5.

[0055] lysosome In this invention, lysosomes are organelles isolated from cells. The structure or composition of lysosomes is not particularly limited as long as they possess the function of decomposing or inactivating viruses. Generally, lysosomes contain hydrolytic enzymes, preferably a combination of multiple hydrolytic enzymes, especially acidic hydrolytic enzymes. Examples include, but are not limited to, proteases, nucleases, phosphatases, glycosidases, lipases, phosphatases, and sulfatases. The inventors have discovered that while the lysosomes of this invention can decompose or inactivate viruses, they do not hydrolyze virus-binding molecules such as AEC2.

[0056] In this invention, lysosomes can be obtained from cell isolation or through artificial synthesis. The method of obtaining lysosomes is not limited as long as they possess the function of decomposing or inactivating viruses. Therefore, lysosomes can be naturally occurring lysosomes, modified lysosomes that have undergone further modification or alteration, or completely artificially synthesized lysosomes.

[0057] In some embodiments, the lysosomes of the present invention are directly isolated from cells. The cell source for the lysosomes is not particularly limited and can be any cell source. Generally, eukaryotic cells are used, preferably immune cells such as macrophages, examples of which include, but are not limited to, primary macrophages, M1 macrophages, and engineered macrophages. The present invention may use any one of the above-mentioned cells, or a combination of two or more. In some embodiments, the cells used in the present invention are engineered macrophages, which are genetically engineered to overexpress viral receptor molecules.

[0058] In some embodiments, the lysosomes of the present invention are obtained through artificial synthesis. For example, lysosomes are obtained by encapsulating a complex hydrolytic enzyme within microspheres prepared from an artificial membrane material. The components of the artificial membrane material include, but are not limited to, phospholipids, neutral lipids, and amphoteric lipids. Examples of phospholipids include, but are not limited to, natural phospholipids, sphingolipids, glycosphingolipids, phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, phosphatidylinositol, and sphingomyelin, or synthetic phospholipids such as dipalmitoylphosphatidylcholine, serine, or ethanolamine glycerol, or the corresponding oleate esters of these compounds. Examples of neutral lipids include, but are not limited to, dioleoylphosphatidylethanolamine (DOPE), cholesterol, and methoxy(polyethylene glycol)-distearate mPEG-DS. Examples of amphoteric lipids include, but are not limited to, DPPG.

[0059] Preparation method A second aspect of the present invention provides a method for preparing a lysosomal preparation, sometimes referred to herein simply as "the preparation method of the present invention", which includes the step of binding virus-binding molecules to the surface of lysosomes, or includes the step of generating virus-binding molecules from isolated lysosomes.

[0060] In some embodiments, the preparation method of the present invention is a chemical modification method, which includes a lysosome isolation step and a chemical modification step of the isolated lysosomes. The lysosome isolation step includes a cell disruption to obtain a mixture, and a step of separating and enriching lysosomes from the mixture. In this document, cell disruption can be performed by known methods, including but not limited to homogenization, sonication, and grinding. Lysosome isolation or enrichment includes centrifugation, such as differential centrifugation. Next, the isolated lysosomes are chemically modified to bind, immobilize, or generate virus-binding molecules. Exemplarily, chemical modification includes adding linker molecules to the lysosome solution for co-incubation; optionally, a further step of separating free linker molecules is performed. In another exemplary embodiment, chemical modification includes adding sialyl cytidine monophosphate and sialyl transferase to the lysosome solution and co-incubating. The incubation temperature is not limited, but is generally 20-40°C, such as 30°C, 37°C, etc.

[0061] In some embodiments, the preparation method of the present invention is a genetic engineering method, which includes the steps of overexpressing a viral binding molecule or its precursor in cells and isolating lysosomes. The step of overexpressing the viral binding molecule or its precursor in cells includes constructing a vector expressing the gene of the viral binding molecule or its precursor, transferring the vector into cells, and culturing them to produce cells containing the viral binding molecule or its precursor. Preferably, the viral binding molecule or its precursor is fused with a lysosomal localization signal peptide, such as LAMP or a fragment thereof, through genetic engineering. In this document, examples of vectors are not limited, but generally include a target gene encoding the viral binding molecule or its precursor, a promoter, a terminator, or optionally, a marker gene. Known vectors or self-constructed vectors can be used. Known vectors include plasmid vectors, lentiviral vectors, adenovirus vectors, AAV viral vectors, etc. In this embodiment, the step of isolating lysosomes can be the same as the chemical modification method, and will not be described again here.

[0062] use A third aspect of the invention provides the use of a combination of a lysosome and a precursor for generating a virus-binding molecule on the surface of the lysosome in the preparation of a medicament for preventing and controlling viral infections.

[0063] In this invention, the form of the drug for preventing and controlling viral infection is not limited and may include any known product. Exemplary examples include products for clearing viruses from a patient's body, products for preparing viral protection, products for clearing influenza virus from a patient with influenza virus, and products for influenza virus protection, etc.

[0064] In this invention, prevention and control include reducing or decreasing the viral load in the subject's body, or clearing the virus from the subject's body, or reducing or weakening the viral activity in the subject's body.

[0065] In this invention, the medicament may further comprise other components, such as other active ingredients or pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers are well known in the art and can be determined by those skilled in the art to meet clinical criteria. Pharmaceutically acceptable carriers include diluents and excipients. Examples of suitable pharmaceutically acceptable carriers include, but are not limited to: (1) Dulbecco phosphate buffered solution, pH about 7.4, containing or not containing about 1 mg / ml to 25 mg / ml human serum albumin; (2) 0.9% saline (0.9% w / v sodium chloride), 5% (w / v) glucose; (3) may also contain antioxidants such as tryptophan and stabilizers such as Tween20.

[0066] The pharmaceutical composition of the present invention can be any suitable dosage form. For example, it can be an injection, a suspension, an emulsion, etc. The pharmaceutical composition of the present invention can be administered into the body in a known manner. For example, it can be delivered to the tissue of interest by intramuscular injection, or optionally by intravenous, percutaneous, intranasal, oral, mucosal, or other delivery methods. Such administration can be performed by a single dose or multiple doses. It will be understood by those skilled in the art that the actual dose to be administered herein can vary considerably depending on a variety of factors, such as the target cells, the biological type or tissue thereof, the general condition of the subject to be treated, the route of administration, the manner of administration, etc.

[0067] In a preferred embodiment, the drug of the present invention is administered via pulmonary aerosol injection, exhibiting excellent bioavailability and superior biocompatibility. Pulmonary aerosol injection is particularly suitable for respiratory-associated viruses. For example, in the lungs, antiviral lysosomal preparations can efficiently clear viruses by first capturing the virus, then internalizing it to completely isolate it from host cells, and finally degrading the virus using lysosomal hydrolases, reducing the possibility of escape or persistent infection. Example 1

[0068] This embodiment is an exemplary example of the preparation of an antiviral lysosomal preparation.

[0069] 1. In vitro culture of primary macrophages and extraction of lysosomes Primary macrophages were obtained from the peritoneal cavity of mice. First, a 6% starch broth solution was prepared: 0.03 g of beef extract, 0.1 g of peptone, 0.05 g of sodium chloride, and 0.6 g of soluble starch were weighed and placed in a small beaker, and 10 mL of ultrapure water was added. The mixture was then heated in a microwave oven until clear and transparent. Next, 5- to 7-week-old C57BL / 6 black mice were selected, and each mouse was injected intraperitoneally with 1 mL of the above starch broth solution. Two days later, all mice were euthanized by cervical dislocation. The mice were then fixed to a foam board, exposing their abdomens. Each mouse was injected intraperitoneally with 5 ml of pre-cooled sterile PBS, and the abdomen was massaged for three minutes, then shaken up and down to detach peritoneal macrophages. The skin was cut open, and the ascites was carefully aspirated with a syringe. The peritoneum was then cut open, and the remaining ascites was aspirated with a pipette. After complete collection, the cells were centrifuged (1500 rpm, 5 min), the supernatant was discarded, and 3 mL of erythrocyte lysis buffer was added to lyse the erythrocytes. The cells were then centrifuged (1500 rpm, 5 min), the supernatant was discarded, and the cells were washed with sterile PBS by centrifugation to obtain primary peritoneal macrophages.

[0070] The obtained primary macrophages were seeded in 6-well plates and incubated statically at 37°C in a 5% CO2 incubator. After 1 day, all supernatant was aspirated, and prepared 1640 medium (10% fetal bovine serum + 1% penicillin-streptomycin + LPS (1 ng / ml) + 1640 medium) was added and incubated in a constant temperature incubator. After 2 days of stimulation, the primary macrophages polarized into M1 macrophages.

[0071] After the primary macrophages were successfully polarized into M1 cells, all primary macrophages were collected. The primary macrophages were homogenized and cell debris was removed by differential centrifugation to enrich lysosomes. Their morphology was observed by transmission electron microscopy (TEM), and their particle size and potential were measured using a nanoparticle tracking analyzer (NTA).

[0072] 2. Preparation of antiviral lysosomal preparations using chemical modification methods DSPE-PEG-NTA-Ni (the optimal mass ratio of lysosomes to DSPE-PEG-NTA-Ni is 50:1) was added to the lysosomal solution obtained above and incubated at 37°C for 1 hour. Then, the free DSPE-PEG-NTA-Ni was separated by ultracentrifugation and the lysosomes were resuspended. The lysosomes obtained above were mixed with ACE2 protein at a protein mass ratio of 20:1 and incubated at 37°C for 1 hour. The free ACE2 protein was then separated by ultracentrifugation, and the antiviral lysosomal preparation was successfully prepared.

[0073] 3. Preparation of antiviral lysosomal preparations using genetic engineering techniques. The macrophages obtained above were incubated with a lentiviral vector expressing ACE2 protein particles and placed in a 37°C incubator containing 5% CO2. After 2 days, all supernatant was aspirated, and 1640 medium (10% fetal bovine serum + 1% penicillin-streptomycin + LPS (1 ng / ml) + 1640 medium) was added and incubated in a constant temperature incubator. After 2 days of stimulation, the primary macrophages polarized into M1 macrophages, and lysosomal preparations were obtained using the lysosomal extraction method described above.

[0074] 4. Characterization of lysosomes The obtained lysosomes and lysosomal preparations were sequentially mixed with ACE2 protein fluorescent antibodies and LAMP-2 protein fluorescent antibodies, and observed under a confocal microscope. The results showed that the fluorescence signals of the lysosomal marker LAMP in the lysosomal preparation group and the fluorescence signals of the ACE2 protein group were highly colocalized, indicating successful ACE2 protein modification. Further detection of the main protein components of the lysosomes and lysosomal preparations using Western blotting showed that both LAMP-2 and ACE2 proteins were positive in lysosomal preparations prepared by chemical modification or genetic engineering modification. Figure 1 and Figure 2 ).

[0075] The morphology of lysosomes and lysosomal formulations was observed using TEM, and the particle size and potential of lysosomes and lysosomal formulations were measured using a nanoparticle tracking analyzer. The results showed that the surfaces of lysosomes and lysosomal formulations were smooth and essentially spherical. The particle size of lysosomes was approximately 467 nm with a potential of -37.6 mV, while the particle size of lysosomal formulations was approximately 492 nm with a potential of -33.8 mV. No significant differences were observed in the physicochemical properties of lysosomes before and after ACE2 protein modification. Figure 3 and Figure 4 ).

[0076] The enzyme activities of ACE2 and lysosomes before and after modification were detected by ACE2 enzyme activity assay, protease enzyme activity assay, and RNase enzyme activity assay. The results showed that ACE2 protein modification did not affect the activity of ACE2 protease, protease, and RNase in lysosomes. Figure 5 ). Example 2

[0077] This example demonstrates the in vitro anticoronavirus efficacy of lysosomal preparations.

[0078] 1. Lysosomal preparations can effectively capture pseudoviruses. The capture ability of lysosomal preparations against pseudoviruses was detected using a quartz electronic microbalance. 100 μg of lysosomes and lysosomal preparations were suspended on the chip surface of the quartz electronic microbalance. A diluted pseudovirus solution was then passed through the chip, and the instrument collected the electron signals transmitted from the chip. The results showed that compared to lysosomes alone, the lysosomal preparations significantly improved the capture ability against pseudoviruses. Figure 6 ).

[0079] 2. Lysosomal preparations can effectively internalize pseudoviruses. The pseudovirus and lysosomal preparation were mixed at a ratio of 5:1 and incubated at 37°C for 1 hour. The solution was then centrifuged using ultracentrifugation to obtain a precipitate of the pseudovirus and lysosomal preparation mixture. The precipitate was then fixed, and electron microscopy sections of the mixture were prepared using a cryo-microtome and observed by TEM. The results showed that four pseudoviruses were present inside the lysosomes, indicating that the lysosomal preparation successfully internalized the pseudoviruses. Figure 7 The two methods are respectively: one is to prepare lysosomes by dropping them onto a copper grid; and the other is to prepare lysosomes by making electron microscopy sections and then placing the sections on a copper grid for observation.

[0080] 3. Lysosomal preparations can degrade pseudoviruses. The pseudovirus and lysosomal preparation were mixed at a ratio of 5:1 and incubated at 37°C. The structural proteins P55 and P24 of the pseudovirus were detected using ELISA; the green fluorescent protein RNA component within the pseudovirus was detected using qPCR. It was observed that the viral proteins and nucleic acid components were slowly degraded, and the protein and nucleic acid signals of the pseudovirus essentially disappeared after 24 hours. Figure 8 and Figure 9 ).

[0081] 4. Lysosomal preparations inhibit pseudovirus infection of host cells. Approximately 10 small focused inoculation dishes were used. 4 One ACE2-OE 293T cell was cultured in an incubator for 24 hours, and then 2×10⁻⁶ cells were added. 4 PFU of pseudoviruses and 10 μg of lysosomal preparation were used to treat the cells for 24 hours. The intensity of green fluorescent protein expressed during pseudovirus infection was observed using confocal microscopy. Two control groups were set up: lysosome-only and ACE2-only, as well as a PBS control group (the number of lysosomes in the lysosomal preparation group was the same as in the lysosomal preparation group; the number of ACE2 molecules in the ACE2-only group was the same as in the lysosomal preparation group). Results showed that, compared to other groups, the lysosomal preparation group significantly inhibited pseudovirus infection at the cellular level. Figure 10 ).

[0082] Approximately 10 seeds were inoculated into each well of the 24-well plate. 4 One ACE2-OE 293T cell was cultured in an incubator for 24 hours, and then 2×10⁻⁶ cells were added.4 Cells were treated with 10 μg of lysosomal preparation and 10 pF of pseudovirus for 24 hours. Cells were collected, and the proportion of cells infected with pseudovirus was analyzed by flow cytometry. Two control groups were set up: one with lysosomes alone and one with ACE2 protein alone, as well as a PBS control group (the number of lysosomes in the lysosomal preparation group was the same as in the lysosomal preparation group; the number of ACE2 cells in the ACE2 protein alone group was the same as in the lysosomal preparation group). Results showed that compared with other groups, the lysosomal preparation group significantly inhibited pseudovirus infection at the cellular level, reducing the proportion of infected cells from 90% to 1%. Figure 11 ). Example 3

[0083] This example illustrates the in vivo distribution and anti-coronavirus effect of lysosomal preparations in mice.

[0084] 1. Distribution of lysosomal preparations in mice Lysosomes were labeled with Cy7-NHS, and then the Cy7-labeled lysosomes were prepared into a lysosomal formulation according to the preparation method in Example 1. Appropriate mice were randomly divided into two groups. One group received 100 μg / mouse of the Cy7-labeled lysosomal formulation via tail vein injection, while the other group received 100 μg / mouse of the Cy7-labeled lysosomal formulation via lung spray. Fluorescence imaging of the mice was then observed in real time using a small animal imaging system. Results showed that, compared to tail vein injection, lung spray administration significantly increased the accumulation of the lysosomal formulation in the lungs. Figure 12 Mice were euthanized by cervical dislocation, and their heart, liver, spleen, lungs, and kidneys were dissected and photographed using a small animal imaging system. Results showed that the vast majority of lysosomal preparation particles administered via pulmonary nebulization accumulated in the lungs. Figure 13 ).

[0085] 2. Safety of lysosomal preparations C57 mice were administered lysosomal preparations via lung spray at a dose of 100 μg / mouse. Three days later, blood samples were collected from the orbital region of the mice for routine blood tests. Two control groups were established: one for lysosomes alone and one for ACE2 protein alone, as well as a PBS control group. The results showed that after lung spray administration of the lysosomal preparations, there were no significant fluctuations in the numbers of red blood cells, platelets, and white blood cells. Figure 14 ).

[0086] Three days later, a suitable amount of blood was collected from the orbital cavity of each administered mouse, and serum was obtained by centrifugation for comprehensive biochemical analysis. The results showed that after lysosomal preparation administration via pulmonary spray, all biochemical indicators were within the normal range. Figure 15 ).

[0087] Three days later, the mice were euthanized, and their internal organs (heart, liver, spleen, lungs, and kidneys) were collected, fixed, embedded in paraffin, and sectioned to 3-4 μm / slice. These sections were stained with hematoxylin and eosin (HE) and observed under a light microscope. Results showed that no significant abnormalities were observed in the internal organs, especially the lungs, after lysosomal preparation administration via pulmonary spray. Figure 16 ).

[0088] 3. The effect of lysosomal preparations on clearing coronaviruses in mouse models A mouse model was established by expressing human ACE2 protein in mouse lung tissue using transgenic methods; subsequently, appropriate hACE2 mice were selected, and each mouse was directly injected with 2×10⁻⁶ ACE2 protein. 6 One hour later, mice were administered Cy7-labeled lysosomal preparations at a dose of 100 μg / mouse via lung spray. Forty-eight hours later, some mice were euthanized by cervical dislocation, and lung tissue was dissected, embedded, fixed, and frozen sectioned. After DAPI staining, the expression of green fluorescent protein in the lungs during viral infection was observed under a microscope. Two control groups were set up: one for lysosomes alone and one for ACE2 protein alone, as well as a PBS control group (the number of lysosomes in the lysosomal preparation group was the same as in the lysosomal preparation group; the number of ACE2 proteins in the ACE2 protein alone group was the same as in the lysosomal preparation group). Results showed that, compared to other groups, the lysosomal preparation group significantly inhibited viral infection at the animal level. Figure 17 ).

[0089] Following the same steps, Omicron virus infection studies were conducted in mouse models. The results showed that the clearance capacity of the lysosomal preparation group was not affected, and it still significantly inhibited the virus infection in hACE2 mice. Figure 18 ). Example 4

[0090] This example demonstrates the anti-coronavirus effect of lysosomal preparations on human lung organoids. 1. Construction of human lung organoids Perinatal tissue was taken from the edge of lung cancer tissue after surgical resection in lung cancer patients. This tissue was then enzymatically digested into single cells. After culturing for 1 week, all cells were collected and resuspended in a mixture of lung organoid culture medium and Matrigel (9:1 ratio). 300 μl of the mixture was seeded into each well of a 24-well plate. After solidification, 300 μl of lung organoid culture medium was added to each well. The medium was changed every 3 days. After 2 weeks, when the lung organoids reached approximately 200 μm in size, they were passaged.

[0091] 2. Anti-coronavirus effects of lysosomal preparations on human lung organoids Cultured lung organoids were digested into cell clusters and seeded into 96-well plates at a density of 500 cells per well. After one week of culture, 200 TCID50 virus and 10 μg of lysosomal preparation were added. After 24 hours, a portion of the lung organoids were harvested, RNA was extracted, and the viral RNA copy number was analyzed. Two control groups were set up: one with EIDD-2801 and the other with RBD antibodies, and a PBS control group (the number of EIDD-2801 and RBD antibodies corresponded to the number of ACE2 proteins on the surface of the lysosomal preparation). Results showed that the viral copy number was lower in the lysosomal preparation group compared to conventional antiviral drugs. Figure 19 ).

[0092] Twenty-four hours later, organoids were harvested and subjected to immunofluorescence staining for the coronavirus S protein, then observed under a confocal microscope. The results showed that, compared to conventional coronavirus drugs, the lysosomal preparation significantly inhibited viral S protein expression in organoids, while the lysosomal preparation group showed virtually no S protein signal. Figure 20 ).

[0093] Following the same steps, Omicron virus infection studies were then conducted on human lung organoid models. The results showed that the viral copy number was also lower in the lysosomal preparation group compared to conventional coronavirus drugs; the S protein was also almost not expressed in the lung organoids of the lysosomal preparation group. Figure 21 and 22 ).

[0094] Simultaneously, standard score analysis and cluster analysis were performed on the above data. The results showed that, across all indicators, the antiviral efficacy of the lysosomal formulation group was superior to that of EIDD-2801 and RBD antibodies; furthermore, the efficacy of EIDD-2801 and RBD antibodies differed against both wild-type and Omicron-type viruses, while this was not observed in the lysosomal formulation group. This indicates that rapid viral mutations do not significantly affect the efficacy of the constructed lysosomal formulation. Figure 23 ). Example 5

[0095] This example demonstrates the antiviral efficacy of lysosomal preparations against influenza viruses in mice. 1. Preparation of anti-influenza virus lysosomal preparations using chemical modification methods Primary macrophages were obtained from the peritoneal cavity of mice according to the steps in Example 1, and were cultured in vitro to polarize them into M1 type primary macrophages. Lysosomes were then extracted. The obtained lysosomes were mixed with sialyl cytidine monophosphate at a mass ratio of 200:1, and an appropriate amount of sialyl transferase was added. The mixture was then incubated at 37 degrees Celsius for 1 hour.

[0096] 2. Preparation of anti-influenza virus lysosomal preparations using genetic engineering methods Lentiviral vectors carrying sialyl transferase plasmids were added to the obtained macrophages and incubated statically at 37°C in a 5% CO2 incubator. After 2 days, all supernatant was aspirated, and 1640 medium (10% fetal bovine serum + 1% penicillin-streptomycin + LPS (1 ng / ml) + 1640 medium) was added and incubated in a constant temperature incubator. After 2 days of stimulation, primary macrophages polarized into M1 type macrophages, and lysosomal preparations were obtained using the above-described lysosomal extraction method. The obtained lysosomes and anti-influenza virus lysosomal preparations were sequentially mixed with elderberry-FITC and LAMP-2 protein fluorescent antibodies and observed under a confocal microscope. The results showed that, regardless of whether the lysosomal preparations were prepared by chemical modification or genetic engineering modification, the fluorescence signal of the lysosomal marker LAMP2 was highly colocalized with the fluorescence signal of sialic acid, indicating that the sialic acid modification was successful. Figure 24 and Figure 25 ).

[0097] 3. Antiviral efficacy of anti-influenza virus lysosomal preparations in mice C57 mice were anesthetized by intraperitoneal injection of 0.5% (v / v) pentobarbital (0.1 ml / 10 gbw) and inoculated with 1000 TCID50 of influenza virus via nasal drop. At 2 hours and 2 days, an anti-influenza virus lysosomal preparation was administered via pulmonary spray at a dose of 100 μg / mouse. The weight of the mice was observed and recorded daily after inoculation. A PBS group and a non-infected control group were also included. Results showed that compared to the PBS group, the mice in the anti-influenza virus lysosomal preparation group experienced a rapid decrease in body weight, while the weight of the mice in the anti-influenza virus lysosomal preparation group remained essentially unchanged, showing little difference from the non-infected control group. Figure 26 ).

[0098] Five days later, all mice were euthanized by cervical dislocation. Lung tissue was dissected and removed, the moisture in the lung tissue was blotted dry with filter paper, and the tissue was weighed. Results showed that the lung index in the anti-influenza virus lysosomal preparation experimental group was lower than that in the PBS group, and essentially consistent with the non-infected control group. Figure 27 RNA was extracted from the lung tissue, and the influenza virus nucleic acid copy number was detected by q-PCR. The results showed that the influenza virus nucleic acid copy number in the anti-influenza virus lysosomal preparation experimental group was significantly lower than that in the PBS group. Figure 28 ).

[0099] Although the invention has been described with reference to exemplary embodiments, it should be understood that the invention is not limited to the disclosed exemplary embodiments. Various adjustments or changes may be made to the exemplary embodiments described in this specification without departing from the scope or spirit of the invention. The scope of the claims should be interpreted in the broadest possible sense to cover all modifications and equivalent structures and functions.

Claims

1. A lysosome-based antiviral agent, characterized in that, The antiviral agent comprises lysosomes and virus-binding molecules located on the surface of the lysosomes, wherein the lysosomes are derived from macrophages, and the virus-binding molecules include receptors for viral surface proteins, wherein the receptors are angiotensin-converting enzyme 2. The antiviral agent is prepared by chemical modification or genetic engineering modification, wherein the chemical modification includes adding DSPE to the lysosomal solution. PEG NTA Ni, mixed with ACE2 protein, wherein the genetic engineering modification includes adding a lentiviral vector carrying ACE2 protein particles to macrophages, and the antiviral agent is characterized to be positive for both LAMP-2 and ACE2 proteins; or The antiviral agent comprises lysosomes and a precursor for producing a virus-binding molecule. The lysosomes are derived from macrophages. The virus-binding molecule includes a receptor for a viral surface protein, wherein the receptor is sialic acid. The precursor is sialytylcytidine monophosphate and sialyltransferase. The antiviral agent is prepared by chemical modification or genetic engineering modification. The chemical modification includes co-incubating lysosomes with sialytylcytidine monophosphate and sialyltransferase. The genetic engineering modification includes adding a lentiviral vector carrying a sialyltransferase plasmid to macrophages. The antiviral agent is characterized to be positive for both LAMP-2 and sialic acid.

2. The lysosome-based antiviral agent according to claim 1, characterized in that, The macrophages include at least one of primary macrophages, M1 macrophages, and engineered macrophages.

3. The method for preparing the lysosome-based antiviral agent according to claim 1 or 2.

4. The use of lysosomes in the preparation of drugs for the prevention and control of viral infections, characterized in that, The lysosomes are modified to have virus-binding molecules on their surface. The lysosomes are derived from macrophages, and the virus-binding molecules include receptors for viral surface proteins, such as angiotensin-converting enzyme 2 or sialic acid. The lysosomes are modified through chemical or genetic engineering modifications. The chemical modification includes adding DSPE to the lysosomal solution. PEG NTA Ni, mixed with ACE2 protein, wherein the genetic engineering modification includes adding a lentiviral vector carrying ACE2 protein particles to macrophages, and the lysosomes are characterized to be positive for both LAMP-2 and ACE2 protein; or The chemical modification includes co-incubating lysosomes with sialyl cytidine monophosphate and sialyl transferase, and the genetic engineering modification includes adding a lentiviral vector carrying a sialyl transferase plasmid to macrophages. The lysosomes are characterized to be positive for both LAMP-2 and sialic acid.

5. The use of a combination of lysosomes and a precursor for generating virus-binding molecules on the surface of the lysosomes in the preparation of a medicament for the prevention and control of viral infections, characterized in that, The lysosomes are derived from macrophages, and the precursors are sialyl cytidine monophosphate and sialyl transferase. The lysosomes are modified by chemical modification or genetic engineering modification. The chemical modification includes co-incubating the lysosomes with sialyl cytidine monophosphate and sialyl transferase. The genetic engineering modification includes adding a lentiviral vector carrying a sialyl transferase plasmid to macrophages. The lysosomes are characterized to be positive for both LAMP-2 and sialic acid.

6. The use according to claim 4 or 5, characterized in that, The prevention and control measures include: To reduce or eliminate the viral load in the subject's body; or Reduce or weaken viral activity in the subject.

Images