Modified plant virus particle and application thereof in preparation of medicine for inhibiting pulmonary fibrosis

Abstract

The invention discloses a modified plant virus particle and application thereof in preparation of a medicine for inhibiting pulmonary fibrosis. The modified plant virus particles comprise: (a) a recognition element targeting fibrosis-promoting macrophages; (b) a linker; (c) plant viruses; the recognition element targeting the fibrosis-promoting macrophages is connected to the outer surface of the plant virus through a linker. The modified plant virus particle provided by the invention can be used as an exogenous regulatory factor for targeted regulation of over-activated fibrosis-promoting macrophages to inhibit pulmonary fibrosis.

Description

Technical Field

[0001] The present invention relates to the technical field of immunotherapy. More specifically, it relates to a modified plant virus particle for inhibiting pulmonary fibrosis and its applications. Background Art

[0002] Idiopathic pulmonary fibrosis is a chronic, progressive, and destructive lung disease caused by abnormal repair of lung injury, but the cause of the disease is still unclear. Zheng, Q et al. statistically analyzed the age-standardized mortality rate of idiopathic pulmonary fibrosis after 2000 and found that the mortality rate of idiopathic pulmonary fibrosis was 0.5 - 12 per year per 100,000 people (Zheng, Q. et al. Mortality and survival in idiopathic pulmonary fibrosis: a systematic review and meta-analysis. ERJ Open Res. 8, 00591 - 02021 (2022).), and its mortality burden is as serious as that of several cancers such as esophageal cancer and pancreatic cancer. Currently, there are two therapeutic drugs approved by the US Food and Drug Administration (FDA) for idiopathic pulmonary fibrosis: Pirfenidone and Nintedanib. Nintedanib is an intracellular inhibitor of tyrosine kinase and has anti-inflammatory and anti-fibrotic effects; Pirfenidone is a drug with anti-inflammatory and anti-fibrotic properties and can reduce the levels of tumor necrosis factor-α (TNF-α), platelet-derived growth factor (PDGF), and transforming growth factor β (TGF-β), etc. In clinical trials and subsequent clinical practice, both Pirfenidone and Nintedanib can reduce the decline of patients' lung function and lower the early mortality rate and all-cause mortality rate of patients. However, during the treatment process, there are widespread adverse reactions, and the prognosis of patients is still very poor. Therefore, it is necessary to explore new treatment regimens for idiopathic pulmonary fibrosis.

[0003] Macrophages are innate immune cells distributed throughout the body. Under the action of the tissue microenvironment and related cytokines, macrophages are activated into different phenotypes (M1 type and M2 type) to participate in physiological processes such as tissue injury repair. However, over-activated M2-type macrophages will secrete pro-fibrotic cytokines, promote the abnormal proliferation of myofibroblasts and excessive deposition of the extracellular matrix, leading to the occurrence of pulmonary fibrosis, and such macrophages are also called pro-fibrotic macrophages.

[0004] In recent years, the regulation of related cytokines and immune cells has become an emerging treatment strategy for fibrosis-related diseases. For example, monoclonal antibodies are used to target colony-stimulating factor 1 receptor (CSF-1R) to block CSF-1R signal transduction, thereby regulating macrophage differentiation and improving fibrosis (Bajpai, A. et al. Axatilimab ameliorates inflammation and fibrosis by targeting macrophages in a preclinical model of chronic GVHD. Blood 142, 2540 (2023).), and serum amyloid P (SAP) is used to regulate macrophage activation to improve the rate of decline in lung function (Pilling D, Roife D, Wang M, et al. Reduction of bleomycin-induced pulmonary fibrosis by serum amyloid P. J Immunol. 2007; 179(6):4035.). However, in the disease process of fibrosis, multiple cytokines act synergistically, and the effect of targeting only one cytokine is limited; moreover, existing proteins and cytokines in the human body are involved in multiple physiological activities, and excessive regulation may affect other normal physiological activities. Therefore, it is necessary to find an exogenous regulatory factor to regulate immune cells and treat pulmonary fibrosis. Summary of the Invention

[0005] Aiming at the problems existing in the above-mentioned prior art, the purpose of the present invention is to provide a modified plant virus particle, which can be used as an exogenous regulatory factor to target and regulate over-activated pro-fibrotic macrophages (such as M2 macrophages) to inhibit pulmonary fibrosis.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] In the first aspect, the present invention provides a modified plant virus particle, which comprises:

[0008] (a) an identification element targeting pro-fibrotic macrophages;

[0009] (b) a linker;

[0010] (c) a plant virus;

[0011] The identification element targeting pro-fibrotic macrophages is connected to the outer surface of the plant virus through a linker.

[0012] Macrophages are activated into different phenotypes under the stimulation of the tissue microenvironment and related cytokines to participate in physiological processes such as tissue damage repair. Over-activated M2 macrophages (i.e., profibrotic macrophages) secrete profibrotic cytokines, promoting the abnormal proliferation of myofibroblasts and excessive deposition of extracellular matrix, leading to the occurrence and progression of pulmonary fibrosis. The inventors of the present invention have found that some plant viruses can regulate macrophage phenotypes and are non-pathogenic to animals or humans, making them ideal exogenous regulators for macrophages. However, the action range of natural plant viruses is too wide, which may cause side effects. Therefore, the present invention modifies natural plant viruses so that they can target and regulate over-activated profibrotic macrophages, i.e., M2 macrophages, restore macrophage homeostasis to a certain extent, reduce the secretion of profibrotic cytokines, and thus inhibit the activation of myofibroblasts and the process of pulmonary fibrosis.

[0013] Furthermore, in the present invention, the profibrotic macrophages refer to M2 macrophages. According to the specific embodiments of the present invention, the profibrotic macrophages are CD206 + (mannose receptor) M2 macrophages.

[0014] Furthermore, the recognition element targeting profibrotic macrophages is selected from one of mannose, M2 macrophage-targeting peptide (M2pep), trehalose, and N-acetylglucosamine. Among them, the amino acid sequence of the M2 targeting peptide is CYEQDPWGVKWW.

[0015] Furthermore, the linker is polyethylene glycol with a molecular weight of 400 - 5000 Da.

[0016] Furthermore, the plant virus is selected from one of tobacco mosaic virus (TMV), cowpea mosaic virus, and potato virus X.

[0017] In the present invention, the recognition element targeting profibrotic macrophages can be connected to the outer surface of the plant virus through a linker by a method known in the art to obtain modified plant virus particles.

[0018] According to the specific embodiments of the present invention, the modified plant virus particles are modified tobacco mosaic virus particles obtained by connecting mannose to the outer surface of tobacco mosaic virus through polyethylene glycol. The specific preparation method includes:

[0019] Modify the tyrosine residues on the outer surface of tobacco mosaic virus to obtain tobacco mosaic virus modified with alkynyl groups on the outer surface;

[0020] Polyethylene glycol with a carboxyl group at one end and an azide group at the other end was used as a linker, and mannose was linked to the outer surface of tobacco mosaic virus through Click reaction and esterification reaction to obtain the modified tobacco mosaic virus particle TMV-OEG8-Man.

[0021] The mannose receptor CD206 can recognize and bind mannose, enabling TMV-OEG8-Man to target CD206. + Pro-fibrotic macrophage population, and then TMV plays a role in regulating the phenotype of activated macrophages. Adding TMV-OEG8-Man (final concentration of 50 μg / mL) to the macrophage culture system and stimulating for 24 h can, to a certain extent, restore the homeostasis of M2 macrophages; the regulated macrophages can inhibit the activation of α-SMA + (α-smooth muscle actin) myofibroblasts. TMV-OEG8-Man has good biocompatibility, is negatively charged in the physiological environment (pH = 7.41), and the modified virus nanoparticles are still rod-shaped with a length of less than 500 nm, having the potential to penetrate the lung mucus layer. Therefore, it can be administered by local atomization, enabling it to be enriched at the lesion site to the greatest extent and minimizing the burden on other tissues and organs, and inhibiting bleomycin-induced pulmonary fibrosis.

[0022] Furthermore, when other recognition elements targeting pro-fibrotic macrophages are selected, the esterification reaction and click chemistry methods can still be used to link them to tobacco mosaic virus, and the above effects can also be achieved.

[0023] In a second aspect, the present invention provides the use of the above-mentioned modified plant virus particles in the preparation of drugs for inhibiting pulmonary fibrosis. As Figure 1 shown, the present invention uses the plant virus particles modified according to the above technical scheme to treat mice with a pulmonary fibrosis model.

[0024] Furthermore, the pulmonary fibrosis is idiopathic pulmonary fibrosis.

[0025] Furthermore, the dosage form of the drug is an injection or an aerosol.

[0026] The beneficial effects of the present invention are as follows:

[0027] The present invention uses modified plant virus particles to target and regulate pro-fibrotic macrophage populations, enabling over-activated macrophages to restore homeostasis to a certain extent and inhibiting the activation of myofibroblasts, thereby inhibiting the progression of pulmonary fibrosis. At the same time, the modified plant virus particles of the present invention can be locally enriched in the lungs by aerosol administration, effectively reducing the impact on other immune cells and normal physiological processes. Description of the Drawings

[0028] The following further elaborates on the specific implementation manners of the present invention in conjunction with the accompanying drawings.

[0029] Figure 1 Shows a schematic diagram of the administration of a modified plant virus as an anti-pulmonary fibrosis drug in experimental animals in the content of the present invention.

[0030] Figure 2 Shows the synthesis route of the modified tobacco mosaic virus in Examples 1-3 of the present invention. a. Modification of tyrosine residues on the outer surface of TMV and reaction with diazonium salt to modify as alkynyl; b. Esterification reaction of PEG and mannose; c. Click reaction of OEG8-Man and TMV-Alkyne to graft OEG8-Man on the outer surface of TMV.

[0031] Figure 3 Shows the characterization results of the modified tobacco mosaic virus in Example 4 of the present invention. a. SDS-PAGE gel electrophoresis results and band gray-scale analysis of tobacco mosaic virus before and after modification; b. Typical TEM images of tobacco mosaic virus before and after modification, scale bar: 500 nm; c. Zeta potential results of tobacco mosaic virus before and after modification under the conditions of pH = 7.41, concentration 0.1 mg / mL, and solvent being 0.01 M PB buffer.

[0032] Figure 4 Shows the cytotoxicity and blood compatibility results of the modified tobacco mosaic virus in Example 5 of the present invention. a. CCK8 results of the cytotoxicity of different concentrations of TMV-OEG8-Man on primary mouse bone marrow-derived macrophages and primary lung fibroblasts; b. Hemolysis rate of different concentrations of TMV-OEG8-Man.

[0033] Figure 5 Shows that the modified tobacco mosaic virus in Example 6 of the present invention regulates the restoration of homeostasis of M2 macrophages to a certain extent. From left to right are the proportion statistics of resting state (M0), M1 type, M2 type, and M2 / M1 type macrophages in each group. In each small graph, from left to right, the groups are Control group, IL-4 alone treatment group, IL-4 treatment + TMV-OEG8-Man treatment group, and LPS&IFN-γ treatment group.

[0034] Figure 6 Shows the inhibitory effect of macrophages regulated by the modified tobacco mosaic virus on the activation of myofibroblasts in Example 7 of the present invention. (1) Fibroblasts co-cultured with resting macrophages. (2) Fibroblasts co-cultured with M2 macrophages regulated by TMV-OEG8-Man. (3) Fibroblasts co-cultured with M2 macrophages. (4) Fibroblasts cultured alone on a hard substrate. Scale bar: 100 μm.

[0035] Figure 7 Shows the enrichment of the modified tobacco mosaic virus in various organs of mice after being inhaled through atomization in Example 8 of the present invention and its biosafety for the main organs (heart, liver, spleen, kidney). a. Ex vivo imaging of the main organs (heart, liver, spleen, lung, kidney) of mice 24 h and 48 h after atomized administration of Sulfo Cy7-labeled TMV-OEG8-Man; b. H&E section results of the main organs (heart, liver, spleen, kidney) of mice 18 days after administration of TMV-OEG8-Man and 28 days after atomized administration of bleomycin. Scale bar: 300 μm.

[0036] Figure 8 Shows the inhibitory effect of the modified tobacco mosaic virus on bleomycin-induced pulmonary fibrosis in mice in Example 9 of the present invention. a. Pathological and CT imaging results of bleomycin-induced pulmonary fibrosis mice with or without TMV-OEG8-Man treatment, scale bar: 300 μm; b. Results of lung coefficient, right lung hydroxyproline content, and survival rate of mice treated differently after the experiment. Detailed implementation manners

[0037] To illustrate the present invention more clearly, the present invention will be further described below in conjunction with preferred embodiments and drawings. Those skilled in the art should understand that the specific content described below is illustrative rather than restrictive, and should not be used to limit the protection scope of the present invention.

[0038] The materials in the following embodiments are commonly used materials in the art and can be obtained from commercial channels unless otherwise specified. The cell culture conditions in the following embodiments are 37 °C, 5% CO2, and saturated humidity unless otherwise specified.

[0039] The reagents and cell sources used in the embodiments of the present invention are as follows:

[0040] DMEM medium, DMEM / F12 medium, penicillin / streptomycin, and trypsin were purchased from Gibco. Fetal bovine serum (FBS) for culturing primary bone marrow-derived macrophages (BMDMs) was purchased from Gibco. When used, FBS (Gibco) and penicillin / streptomycin were added to DMEM medium at final volume fractions of 10% and 1% respectively. FBS for culturing primary lung fibroblasts was purchased from Hyclone. When used, FBS (Hyclone) and penicillin / streptomycin were added to DMEM / F12 medium at final volume fractions of 15% and 1% respectively.

[0041] BMDMs were induced from bone marrow cells of BALB / c mice or C57BL / 6J mice. The specific method was as follows: After removing the femurs and tibias of the mice, the bone marrow was flushed out with pre-cooled PBS. The bone marrow was collected, and the cells filtered through a 70-μm cell strainer were placed in erythrocyte lysate and left standing at 4 °C for 10 min. The remaining cells were collected by centrifugation and cultured in DMEM containing 20 ng / mL M-CSF, 10% FBS, and 1% penicillin / streptomycin. On the 3rd and 5th days, the old culture medium was discarded and replaced with fresh medium. On the 6th day, mature BMDM cells were obtained.

[0042] Primary lung fibroblasts were extracted from the lung tissues of C57BL / 6J mice. The specific method was as follows: The lung tissues of the mice were lavaged 3 times with pre-cooled PBS, and then the lung tissues were taken out and cut into small pieces about 1 mm 3 in size, placed in 10 mL of pure DMEM / F12 containing 300 U of type I collagenase, and incubated in a water bath at 37 °C for 1 h. The remaining cells and tissue blocks were collected by centrifugation and cultured adherently in DMEM / F12 containing 15% FBS and 1% penicillin / streptomycin. On the 3rd and 5th days, the old culture medium was discarded and replaced with fresh medium. On the 7th day, the adherent tissue blocks were removed for passage, and the second-generation cells were used for the experiment.

[0043] Example 1 Modification of the outer surface of TMV with alkynyl

[0044] A 0.3 M aqueous solution of p-toluenesulfonic acid, 0.68 M 3-ethynylaniline / acetonitrile, and 3 M aqueous solution of sodium nitrite were mixed in a ratio of 16:3:1, and reacted in the dark in an ice-water bath for 1 h to prepare a diazonium salt. TMV was mixed with the diazonium salt in 0.1 M borax / boric acid buffer. Stirred and reacted in the dark in an ice-water bath for 30 min. After the reaction, dialysis was carried out to obtain tobacco mosaic virus (TMV-Alkyne) with an outer surface modified with alkynyl (as Figure 2 shown in a).

[0045] Example 2 Esterification reaction of PEG and mannose

[0046] Sugar solution: Weigh a certain amount of mannose and dissolve it in 0.5 M MES buffer.

[0047] Reaction solution: Weigh a certain amount of N3-PEG8-CH2CH2COOH, NHS, and EDC and dissolve them in 0.5 M MES buffer. The ratio of the three is 1:1-4:2-4, and stir at room temperature for 30 min.

[0048] The sugar solution was mixed with the reaction solution and stirred overnight at room temperature to obtain OEG8-Man (as Figure 2 shown in b).

[0049] Example 3 Click Reaction of OEG8-Man and TMV-Alkyne

[0050] Take a certain amount of TMV-Alkyne and OEG8-Man and mix them in 0.1M PB buffer. Subsequently, add aminoguanidine (final concentration 2 mM), sodium ascorbate (final concentration 2 mM), and copper sulfate (final concentration 1 mM) in sequence. After reacting for 30 minutes at 4 degrees in the dark, add EDTA (final concentration 5 mM) and stir for 5 minutes to end the reaction. Then perform dialysis to obtain modified tobacco mosaic virus particles with targeting effects (such as Figure 2 c) in

[0051] Example 4 Characterization of Modified TMV

[0052] Take a certain amount of sample and mix it with 5× protein loading buffer, and boil for 10 min. Install a 15% SDS-PAGE gel, load the sample, and perform electrophoresis at a voltage of 150 - 200 V. After electrophoresis, stain with Coomassie Brilliant Blue staining solution, and use Fiji to perform gray-scale analysis on the bands to calculate the grafting rate of the sample. After grafting of OEG8 and OEG8-Man, 1 additional band and 2 additional bands appeared relative to TMV respectively, indicating successful modification of TMV. At the same time, the grafting rate of OEG8-Man on TMV was calculated to be 13.7% (such as Figure 3 a) in

[0053] Load TMV and TMV-OEG8-Man on one side of the copper grid with a carbon support film, stain with uranyl acetate, and take typical structural photos of the samples through a transmission electron microscope (TEM). The results show that TMV before and after modification is rod-shaped (such as Figure 3 b) in

[0054] Dilute TMV and TMV-OEG8-Man to a concentration of 0.1 mg / mL with 0.01M PB buffer (pH = 7.41), and then perform zeta potential detection. The results show that the surface of TMV-OEG8-Man is negatively charged (such as Figure 3 c) in

[0055] Example 5 Cytocompatibility and Blood Compatibility of Modified Tobacco Mosaic Virus

[0056] Cytocompatibility of TMV-OEG8-Man:

[0057] Dilute TMV-OEG8-Man to 1, 3, 9, 27, 81, 243 μg / mL with culture medium in a gradient manner.

[0058] The control group is the culture medium without TMV-OEG8-Man.

[0059] A. BMDM cells:

[0060] 1. Extract mouse bone marrow cells and seed them in a 96-well plate at a density of 20,000 cells per well. Then, induce them with M-CSF for 6 days to obtain BMDM cells.

[0061] 2. On the 6th day, change the medium to DMEM medium (containing 10% FBS and 1% penicillin / streptomycin) with different concentrations (0 - 243 μg / mL) of TMV-OEG8-Man, and culture for 24 h under normal conditions (37 °C, 5% CO2, saturated humidity).

[0062] 3. Discard the medium, add DMEM medium containing 10% CCK8, incubate for a certain time, then use an ELISA reader to detect OD450 and calculate the cell viability.

[0063] B. Mouse primary lung fibroblasts:

[0064] 1. Seed mouse primary lung fibroblasts in a 96-well plate at a density of 8,000 cells per well and culture for 24 h under normal conditions (37 °C, 5% CO2, saturated humidity).

[0065] 2. Discard the old medium and change it to DMEM / F12 medium (containing 15% FBS and 1% penicillin / streptomycin) with different concentrations (0 - 243 μg / mL) of TMV-OEG8-Man, and culture for 24 h under normal conditions (37 °C, 5% CO2, saturated humidity).

[0066] 3. Discard the medium, add DMEM medium containing 10% CCK8, incubate for a certain time, then use an ELISA reader to detect OD450 and calculate the cell viability.

[0067] The results showed that in the concentration range of 1 - 243 μg / mL, TMV-OEG8-Man had no cytotoxicity to BMDMs and primary lung fibroblasts (as shown in Figure 4 a)).

[0068] Hemocompatibility of TMV-OEG8-Man:

[0069] 1. Take fresh rabbit whole blood, wash it with 1×PBS, and use the obtained red blood cells to prepare a 5% (v / v) red blood cell suspension with 1×PBS.

[0070] 2. Gradient dilute TMV-OEG8-Man with 1×PBS to concentrations of 1, 3, 9, 27, 81, and 243 μg / mL, respectively.

[0071] 3. Mix an appropriate amount of the sample with an equal volume of the erythrocyte suspension and incubate at 37 °C for 1 h. The 1×PBS group serves as the negative control, and the 0.1% Triton X-100 / 1×PBS group serves as the positive control.

[0072] 4. Centrifuge to obtain the supernatant, measure OD540, and calculate the hemolysis rate.

[0073] The results showed that in the concentration range of 1 - 243 μg / mL, TMV-OEG8-Man did not show hemolytic activity (as shown in b) of Figure 4 .

[0074] Example 6 Regulation of M2 macrophages by TMV-OEG8-Man

[0075] 1. After successful induction of BMDMs cells, use DMEM medium (containing 10% FBS and 1% penicillin / streptomycin) containing 100 ng / mL LPS & 20 ng / mL IFN-γ or 25 ng / mL IL-4, and culture for 24 - 48 h under conventional conditions (37 °C, 5% CO2, saturated humidity) to obtain M1 and M2 macrophages. The negative control group is cultured with fresh DMEM medium (containing 10% FBS and 1% penicillin / streptomycin) to obtain resting macrophages.

[0076] 2. Discard the old medium and replace it with DMEM medium (containing 10% FBS and 1% penicillin / streptomycin) containing 50 μg / mL TMV-OEG8-Man to stimulate M2 macrophages; the other groups are replaced with fresh DMEM medium (containing 10% FBS and 1% penicillin / streptomycin). Culture for 24 h under conventional conditions (37 °C, 5% CO2, saturated humidity).

[0077] 3. After the culture, collect the cells and label the cells with fluorescently labeled anti-CD86 and anti-CD206 antibodies. Finally, use flow cytometry to detect the proportions of resting macrophages (M0, CD86 - CD206 - ), M1 macrophages (CD86 + CD206 - ), and M2 macrophages (CD86 - CD206 + ).

[0078] The results showed that macrophages were activated to M2 macrophages after stimulation with IL-4; compared with the resting macrophages in the control group and the M1 macrophages in the LPS & IFN-γ group, treating M2 macrophages with TMV-OEG8-Man could reduce the proportion of M2 macrophages in the macrophage population, making the proportion of different phenotypic macrophages closer to the steady state (as shown in Figure 5)。

[0079] Example 7: Effects of Modified Tobacco Mosaic Virus-Regulated Macrophages on Fibroblasts

[0080] 1. Obtain bone marrow-derived macrophages (BMDMs) and lung fibroblasts from C57BL / 6J mice. Seed BMDM cells into Transwell chambers suitable for 12-well plates, and seed lung fibroblasts into 12-well plates.

[0081] 2. Add IL-4 to the BMDM medium to a final concentration of 25 ng / mL, and culture for 24 - 48 h under conventional conditions (37 °C, 5% CO2, saturated humidity). For one group of M2 macrophages, replace the medium with that containing TMV-OEG8-Man (final concentration 1 μg / mL), and culture for 24 h under conventional conditions (37 °C, 5% CO2, saturated humidity) to obtain TMV-OEG8-Man-regulated M2 macrophages. For resting BMDMs, only synchronously replace the fresh medium without changing other culture conditions.

[0082] 3. Co-culture BMDMs with primary lung fibroblasts for 96 h under conventional conditions (37 °C, 5% CO2, saturated humidity). Primary lung fibroblasts cultured alone on a rigid substrate serve as a positive control for the activation of fibroblasts into myofibroblasts.

[0083] 4. After the co-culture, perform immunofluorescence staining on primary lung fibroblasts to label α-SMA and the nucleus.

[0084] The results showed that compared with the positive control group and the co-culture group with M2 macrophages, the expression of the myofibroblast marker α-SMA in the co-culture group with TMV-OEG8-Man-regulated M2 macrophages was significantly downregulated, approaching that of the co-culture group with resting macrophages, and the activation of myofibroblasts was inhibited (as Figure 6 )。

[0085] Example 8: Enrichment of TMV-OEG8-Man in Mice after Nebulized Administration and Its Biosafety in Mice

[0086] Sulfo Cy7 was labeled on the inner surface of TMV-OEG8-Man through amidation reaction. TMV-OEG8-Man, Sulfo Cy7 NH2 (2 - 10 fold equivalents relative to TMV-OEG8-Man), and HOBT (50 fold equivalents relative to TMV-OEG8-Man) were added into 0.1 M HEPES buffer, and EDC (a total of 90 fold equivalents relative to TMV-OEG8-Man) was added in three portions within the subsequent 24 h. The mixture was stirred at 2 - 8 °C for 24 h. After the reaction, dialysis was carried out to obtain fluorescently labeled TMV-OEG8-Man.

[0087] The fluorescently labeled TMV-OEG8-Man was nebulized into the trachea of mice, and the main organs of the mice (heart, liver, spleen, lung, kidney) were taken out and observed for the enrichment of TMV-OEG8-Man in the mice at 24 h and 48 h respectively. The results showed that after 48 h of entering the body, TMV-OEG8-Man mainly accumulated in the lungs, a small amount entered the kidneys, and no fluorescence was observed in the heart, liver, and spleen (as shown in Figure 7 a) in

[0088] On day 0, mice were nebulized with bleomycin sulfate solution to establish a pulmonary fibrosis mouse model. On day 10, mice were nebulized with TMV-OEG8-Man (dose: 0.1 mg / kg), and the main organs were taken on day 28 to prepare H&E pathological sections. The sham operation group was used as a negative control and nebulized with an equal volume of solvent (i.e., 0.01 M PB buffer, pH = 7.41) on day 0 and day 10. No obvious lesions were observed in the main organs of the mice after administration of TMV-OEG8-Man, indicating that TMV-OEG8-Man has good biosafety (as shown in Figure 7 b) in

[0089] Example 9 Inhibitory effect of TMV-OEG8-Man on bleomycin-induced pulmonary fibrosis in mice

[0090] On the 0th day, mice were nebulized with bleomycin sulfate solution to establish a mouse model of pulmonary fibrosis. On the 10th day, mice were nebulized with TMV-OEG8-Man (at a dose of 0.1 mg / kg) for treatment. The bleomycin group was nebulized with an equal volume of solvent (i.e., 0.01 M PB buffer, pH = 7.41) on the 10th day as a positive control, and the sham operation group was nebulized with an equal volume of solvent (i.e., 0.01 M PB buffer, pH = 7.41) on the 0th day and the 10th day as a negative control. On the 28th day, the mice were weighed and subjected to CT imaging, and then the experiment was terminated. The mouse lung tissues were removed and weighed. The left lung was used for making H&E and Masson pathological sections, and the right lung was used for detecting the hydroxyproline content. The lung coefficient results were calculated based on the wet weight of the mouse lung tissue and the body weight. The experimental results showed that compared with the untreated mice, TMV-OEG8-Man increased the survival rate of the mice and decreased the lung coefficient and the hydroxyproline content in the right lung (as shown in Figure 8 b). In addition, the pathological section and CT results showed that after treatment with TMV-OEG8-Man, the deposition of collagen fibers in the lungs of the mice decreased and the lesions were alleviated, indicating that the symptoms of pulmonary fibrosis in the mice were inhibited. (As shown in Figure 8 a)

[0091] Obviously, the above-mentioned embodiments of the present invention are merely examples for clearly explaining the present invention, and are not intended to limit the embodiments of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the embodiments here. Any obvious changes or modifications derived from the technical solutions of the present invention still fall within the protection scope of the present invention.

Claims

1. A modified plant virus particle, characterized in that, The modified virus particles include: (a) An identification element targeting pro-fibrotic macrophages; (b) A linker; (c) A plant virus; The identification element targeting pro-fibrotic macrophages is connected to the outer surface of the plant virus through a linker.

2. The modified plant virus particle according to claim 1, wherein, The pro-fibrotic macrophages are M2 macrophages.

3. The modified plant virus particle according to claim 1, wherein The identification element targeting pro-fibrotic macrophages is selected from one of mannose, M2 macrophage targeting peptide, trehalose, and N-acetylglucosamine.

4. The modified plant virus particle according to claim 1, characterized in that, The linker is polyethylene glycol with a molecular weight of 400 - 5000 Da.

5. The modified plant virus particle according to claim 1, wherein The plant virus is selected from one of tobacco mosaic virus, cowpea mosaic virus, and potato virus X.

6. The modified plant virus particle according to claim 1, wherein The modified plant virus particles are modified tobacco mosaic virus particles obtained by connecting mannose to the outer surface of tobacco mosaic virus through polyethylene glycol.

7. The modified plant virus particle according to claim 6, wherein The specific method for connecting mannose to the outer surface of tobacco mosaic virus through polyethylene glycol includes: Modifying tyrosine residues on the outer surface of tobacco mosaic virus to obtain tobacco mosaic virus with an alkyne-modified outer surface; Using polyethylene glycol with a carboxyl group at one end and an azide group at the other end as a linker, and connecting mannose to the outer surface of tobacco mosaic virus through Click reaction and esterification reaction.

8. Use of the modified plant virus particles according to any one of claims 1 - 7 in the preparation of a drug for inhibiting pulmonary fibrosis.

9. The application according to claim 8, wherein, The pulmonary fibrosis is idiopathic pulmonary fibrosis.

10. The application according to claim 8, wherein, The dosage form of the drug is an injection or an aerosol.

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