Antiviral nano medicament NM4 as well as preparation method and application thereof

Abstract

The invention discloses an antiviral nano-agent NM4 as well as a preparation method and application thereof, and belongs to the technical field of nano-agent preparation. The anti-virus nano medicament NM4 prepared by the invention has an excellent prevention and treatment effect on plants infected with soybean mosaic viruses, and by applying the medicament NM4 to the plants infected with SMV viruses, the resistance of the soybean plants to SMV infection is obviously enhanced, and the medicament NM4 can inhibit virus accumulation, relieve oxidative stress, protect a cell membrane system and promote photosynthetic metabolism in a synergistic manner, so that the anti-virus nano medicament NM4 has an excellent prevention and treatment effect on the plants infected with the soybean mosaic viruses. Excellent disease-resistant potential and a biological stimulation function are shown. Meanwhile, through multiple mechanisms such as electrostatic adsorption, ROS (reactive oxygen species) regulation, immune activation and energy metabolism maintenance, effective inhibition on SMV infection is jointly realized, and theoretical support is provided for research, development and application of antiviral nano-drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanopharmaceutical preparation, and in particular relates to an antiviral nanopharmaceutical NM4 and a preparation method and application thereof. Background Art

[0002] Soybean mosaic virus (SMV), the largest genus of plant pathogenic viruses in the Potyviridae family, causes soybean leaf mosaic disease or necrosis, stunted plant development, and a mottled seed coat. Under natural field conditions, this can lead to an average annual soybean yield reduction of 15%-40%, and in severe cases, even crop failure. The impact of SMV extends beyond direct field yield reductions to multiple chain reactions, such as reduced crop quality and limited storage, severely hindering the development of my country's soybean industry. SMV's strong adaptability to the environment and seed-transmitted nature make it extremely difficult to control, and there is currently a lack of specific drugs or other effective chemical control methods for SMV.

[0003] In the research journey of viral prevention and treatment, nanomaterials, leveraging their extraordinary physicochemical properties, have become a frontier in the development of novel antiviral agents. Based on their mechanisms of action and application scenarios, nanopharmaceutical systems can be broadly categorized into three categories: 1. Virus-neutralizing nanomaterials: These target direct action on viral particles, inactivating them through physical binding or chemical catalysis. 2. Vector-delivery nanosystems: These significantly improve drug delivery efficiency, enabling superior therapeutic efficacy with reduced drug doses. 3. Immune-stimulating nanoparticles: These focus on activating the host immune system, indirectly exerting their antiviral effects. However, during viral infection, excessive accumulation of reactive oxygen species (ROS) within host cells becomes a key driver of viral replication. Viruses can hijack the ROS mechanism. For example, influenza viruses can activate NADPH oxidase 2 (Nox2) to generate large amounts of ROS, triggering an oxidative stress storm. This not only compromises the host cell's defenses but also promotes viral replication and spread.

[0004] Existing nanotechnology has fallen into a contradictory dilemma when dealing with this problem. On the one hand, ROS-promoting materials (such as iron-based / copper-based nanozymes) rely on the production of ROS to directly destroy viruses, but high concentrations of ROS will aggravate inflammatory damage, forming a vicious cycle of "easy to kill bacteria, difficult to heal"; on the other hand, ROS-scavenging materials (such as cerium-based nanozymes CeTA) can clear ROS and relieve inflammation, but cannot directly block viral replication, and are inefficient due to lack of targeting. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide an antiviral nanoparticle NM4 and a preparation method and application thereof, so as to solve the technical problem that existing drugs have poor control effects on soybean mosaic virus.

[0006] To achieve the above object, the technical solution adopted by the present invention is: to provide a method for preparing an antiviral nanoparticle NM4, comprising the following steps: S1. Dissolve a cobalt salt and an organic ligand in water respectively, and then mix them so that the molar ratio of the cobalt salt to the organic ligand in the mixture is 1.5-2.5:0.5-1.5; then react at 90-110°C for 0.5-1.5 hours to allow the ligand to coordinate with the metal ion to form a stable metal-organic framework structure; then centrifuge, wash, and dry at -35~-30°C for 22-26 hours to obtain Co-MOF (CM); the organic ligand is 2,5-dihydroxyterephthalic acid (DHTA), dimethylimidazole, or NN, dimethylformamide; S2. Mix the manganese salt and alkali solution evenly to obtain solution A, and disperse Co-MOF in water to obtain solution B; then add solution A to solution B, stir and react at room temperature for 10-14 hours, centrifuge, wash, and dry at -35~-30℃ for 22-26 hours to obtain CoMn-MOF (CMM), Mn 2+ The introduction of will form new coordination sites in the framework of Co-MOF, thereby changing the pore structure and specific surface area of ​​MOF; the ratio of manganese salt, alkali solution and Co-MOF is 0.1-0.3mmol:15-25mL:0.3-0.5g; S3. Place CoMn-MOF in a protective atmosphere and heat treat it at 600-800°C for 1-3 hours. At high temperature, the organic ligands in CoMn-MO undergo thermal decomposition to form a carbon layer, and the metal ions are partially reduced to obtain the antiviral nanoparticle NM4 (C@C@MO). The formation of the carbon layer improves the thermal stability of the material, allowing it to maintain good performance in high-temperature environments.

[0007] On the basis of the above technical solution, the present invention can also be improved as follows: Furthermore, the cobalt salt is cobalt acetate or cobalt chloride.

[0008] Furthermore, the manganese salt is manganese chloride or manganese carbonate, and the alkali solution is ammonia water or sodium hydroxide.

[0009] Furthermore, the centrifugal speed in S1 and S2 is 4000-5000 rpm / min, and the time is 3-7 min.

[0010] Furthermore, the washing liquid used in S1 and S2 is water and / or anhydrous ethanol.

[0011] Furthermore, the protective atmosphere is made of nitrogen or argon.

[0012] Furthermore, the heating rate in S3 is 2-5°C / min.

[0013] Furthermore, the heat treatment temperature is 700° C., and the heat treatment time is 3 h.

[0014] The invention also discloses an antiviral nanopharmaceutical NM4 prepared by the preparation method.

[0015] The present invention also discloses the use of the antiviral nanopharmaceutical NM4 in preparing a preparation for treating and / or preventing soybean mosaic virus.

[0016] The beneficial effects of the present invention are: 1. The surface-positively charged (Zeta potential of +10.2-12.1 mV) driving material NM4 prepared by the present invention can specifically aggregate in negatively charged mitochondria / chloroplasts, eliminating virus-induced reactive oxygen species at the source and reducing H2O2 content. The MnO nanoparticles and carbon layer synergistically catalyze the decomposition of ROS, blocking oxidative damage and reducing MDA content. The rough nanorod morphology can enhance biomembrane adsorption, repair virus-infected membrane permeability, and reduce cell membrane conductivity.

[0017] 2. The positive charge of the antiviral nanoparticle NM4 directly adsorbs viral particles / RNA, neutralizing their infectivity. It protects organelle energy synthesis, depriving viruses of the ATP and microenvironment necessary for replication. Furthermore, by repairing membrane lipid integrity, MDA levels decrease, hindering intercellular viral spread. This triple synergistic mechanism of "targeted catalytic clearance, membrane repair, and viral replication blockade" effectively inhibits plant viruses, transcending the limitations of existing antiviral nanoparticles that rely solely on ROS scavenging or passive adsorption, and providing a new paradigm for agricultural nanoantiviral formulations.

[0018] 3. The present invention applied the antiviral nanopharmaceutical NM4 to plants infected with soybean mosaic virus, and found that the resistance of soybean plants to SMV infection was significantly enhanced, which was manifested by improved plant morphology, reduced virus accumulation, and the expression of soybean mosaic virus coat protein gene ( SMV - CPTransmission electron microscopy revealed that treatment with the agent effectively reduced the number of intracellular viral particles and repaired damaged mitochondrial structure. Analysis of physiological indicators further demonstrated that application of the agent NM4 effectively reduced the accumulation of reactive oxygen species in plants, inhibited SMV-induced oxidative stress, and significantly reduced virus-induced oxidative damage to organelles. It also regulated the activity of antioxidant enzymes such as SOD, POD, and CAT, alleviating membrane lipid peroxidation (MDA levels) and cell membrane leakage (conductivity). Furthermore, the treated group showed increased chlorophyll and soluble protein content, and restored photosynthetic capacity and metabolic activity. These findings provide theoretical support for the development and application of antiviral nanomedicines. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 These are scanning electron micrographs of the antiviral nanoparticle NM4; Figure (A) is a SEM image of NM4 at 20x magnification, and Figure (B) is a SEM image of NM4 at 50x magnification; Figure 2 Transmission electron microscopy images of the antiviral nanoparticle NM4; Figure (A) is a high-angle annular bright-field scanning electron microscopy image, Figure (B) is a high-angle annular dark-field scanning electron microscopy image, Figure (C) is a Co element mapping image, Figure (D) is a C element mapping image, Figure (E) is an O element mapping image, and Figure (F) is a Mn element mapping image; Figure 3 XRD patterns of CM, CMM, and NM4; Figure 4 This is the Zeta potential diagram of the antiviral nanoparticle NM4; Figure 5 This is the FTIR spectrum of the antiviral nanoparticle NM4; Figure 6 This is a soybean status map; Figure 7 This is a diagram of soybean leaf mosaic; Figure 8 This is a statistical chart of soybean plant height; Figure 9Electron microscope observations of the intracellular environment; Figure (A) shows the virus status in the leaves of soybean plant A in the CK+ group, Figure (B) shows the virus status in the leaves of soybean plant B in the CK+ group, Figure (C) shows the virus status in the leaves of soybean plant C in the CK+ group, Figure (D) shows the virus status in the leaves of soybean plant A in the NM4 group 4 days after application of the pesticide, Figure (E) shows the virus status in the leaves of soybean plant B in the NM4 group 4 days after application of the pesticide, Figure (F) shows the virus status in the leaves of soybean plant C in the NM4 group 4 days after application of the pesticide, Figure (G) shows the virus status in the leaves of soybean plant A in the NM4 group 7 days after application of the pesticide, Figure (H) shows the virus status in the leaves of soybean plant B in the NM4 group 7 days after application of the pesticide, and Figure (I) shows the virus status in the leaves of soybean plant C in the NM4 group 7 days after application of the pesticide; Figure 10 Figure 1 is a local characteristic diagram of the intracellular environment; Figure (A) shows the local virus status in the leaves of soybean plant A in the CK+ group, Figure (B) shows the local virus status in the leaves of soybean plant B in the CK+ group, Figure (C) shows the local virus status in the leaves of soybean plant A in the NM4 group 7 days after application, and Figure (D) shows the local virus status in the leaves of soybean plant B in the NM4 group 7 days after application; Figure 11 The staining results of NBT and DAB; Figure 12 This is the result of measuring hydrogen peroxide content in soybean leaves; Figure 13 This is the SOD content determination diagram of soybean leaves; Figure 14 This is the POD content determination diagram of soybean leaves; Figure 15 This is the CAT content determination diagram of soybean leaves; Figure 16 This is the MDA content determination diagram of soybean leaves; Figure 17 This is the electrical conductivity measurement diagram of soybean leaves; Figure 18 This is a graph showing the determination of chlorophyll content in soybean leaves; Figure 19 This is a graph showing the determination of soluble protein content in soybean leaves; Figure 20 This is a graph showing the detection of virus replication in soybean leaves; Figure 21 This is a comparison of the virus inhibition effects of the nanoparticle NM4 using different administration methods; Figure 22 The results of qRT-PCR detection of viral content; Figure 23 This is the volcano map of differentially expressed genes in soybeans before and after pesticide application; Figure 24This is the bubble map of GO enrichment of differentially expressed genes before and after pesticide application in soybean; Figure 25 This is the KEGG enrichment analysis diagram; Figure 26 This is a heat map of hormone-related gene expression. DETAILED DESCRIPTION

[0020] The specific embodiments of the present invention are described below to facilitate understanding of the present invention by those skilled in the art. In the examples, where specific conditions are not specified, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used where the manufacturer is not specified are conventional products that can be purchased commercially. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. It is obvious to those skilled in the art that various modifications are within the spirit and scope of the present invention as defined and determined by the appended claims, and all inventions and creations utilizing the present invention are protected.

[0021] Example 1 A method for preparing an antiviral nanoparticle NM4 comprises the following steps: S1. Dissolve 8 mmol of cobalt acetate and 3 mmol of dimethylimidazole in 50 mL of water, then add the cobalt acetate solution to the dimethylimidazole solution and stir for 40 minutes. Then react at 110°C for 0.5 hours. After the reaction, centrifuge at 5000 rpm / min for 3 minutes, wash with water and anhydrous ethanol three times, and freeze-dry at -30°C for 26 hours to obtain Co-MOF. S2. 1.6 mL of sodium hydroxide and 5 mmol of NH4Cl were added to 22 mL of water for ultrasonic dispersion, followed by the addition of 0.3 mmol of manganese carbonate to obtain solution A; 0.3 g of Co-MOF was dispersed in 18 mL of water to obtain solution B; solution A was then added to solution B, and the mixture was stirred at room temperature for 14 h. Finally, the mixture was centrifuged at 5000 rpm / min for 3 min, washed three times with water and anhydrous ethanol, and freeze-dried at -35°C for 22 h to obtain CoMn-MOF; S3. Place the CoMn-MOF in a nitrogen atmosphere, increase the temperature from room temperature to 800°C at a heating rate of 5°C / min, and heat treat for 1 hour to obtain the antiviral nanomedicine NM4.

[0022] Example 2 A method for preparing an antiviral nanoparticle NM4 comprises the following steps: S1. Dissolve 9 mmol of cobalt chloride and 6 mmol of NN, dimethylformamide in 60 mL of water, then add the cobalt acetate solution to the NN, dimethylformamide solution and stir for 20 minutes, then react at 90°C for 1.5 hours. After the reaction, centrifuge at 4000 rpm / min for 7 minutes, wash with water and anhydrous ethanol three times, and freeze-dry at -35°C for 22 hours to obtain Co-MOF. S2. 2 mL of ammonia water and 6 mmol of NH4Cl were added to 18 mL of water and ultrasonically dispersed, followed by the addition of 0.1 mmol of MnCl2˖4H2O to obtain solution A. 0.5 g of Co-MOF was dispersed in 20 mL of water to obtain solution B. Solution A was then added to solution B, and the mixture was stirred at room temperature for 10 h. Finally, the mixture was centrifuged at 4000 rpm / min for 7 min, washed three times with water and anhydrous ethanol, and freeze-dried at -30°C for 26 h to obtain CoMn-MOF. S3. Place the CoMn-MOF in an argon atmosphere, increase the temperature from room temperature to 600°C at a heating rate of 2°C / min, and heat treat for 3 hours to obtain the antiviral nanomedicine NM4.

[0023] Example 3 A method for preparing an antiviral nanoparticle NM4 comprises the following steps: S1. Dissolve 8 mmol of cobalt acetate and 4 mmol of 2,5-dihydroxyterephthalic acid (DHTA) in 60 mL of water, then add the cobalt acetate solution into the DHTA solution and stir for 30 min. Then react at 100 °C for 1 h. Cobalt acetate dissociates in water. 2+ DHTA dissociated from DHTA - The ions underwent coordination reaction; after the reaction, the mixture was centrifuged at 4500 rpm / min for 5 min, washed with water and anhydrous ethanol three times, and freeze-dried at -33°C for 24 h to obtain Co-MOF; S2. 1.6 mL of ammonia water and 6 mmol of NH4Cl were added to 20 mL of water and ultrasonically dispersed, followed by the addition of 0.2 mmol of MnCl2˖4H2O to obtain solution A. 0.4 g of Co-MOF was dispersed in 20 mL of water to obtain solution B. Solution A was then added to solution B, and the mixture was stirred at room temperature for 12 h. Finally, the mixture was centrifuged at 4500 rpm / min for 5 min, washed three times with water and anhydrous ethanol, and freeze-dried at -33°C for 24 h to obtain CoMn-MOF. S3. Place the CoMn-MOF in a nitrogen atmosphere, increase the temperature from room temperature to 700°C at a heating rate of 3°C / min, and heat treat for 2 hours to obtain the antiviral nanomedicine NM4.

[0024] The antiviral nanoparticle NM4 sample used in the following experiments is the NM4 material prepared in Example 3.

[0025] Experimental Example 1 Physical Structure Characterization 1. The shape, size and surface roughness of NM4 particles were analyzed by scanning electron microscopy (SEM). Figure 1 As shown, the framework of the carbon nanorods of C@C@MO contains a large number of uniformly distributed particles.

[0026] 2. The internal structure, particle size uniformity and core-shell structure characteristics of NM4 were observed by transmission electron microscopy (TEM). Figure 2 As shown, the carbon nanorod framework of NM4 contains a large number of evenly distributed particles. Elemental mapping images confirm the presence of the desired elements Co, Mn, and O in the carbon framework.

[0027] 3. Through X-ray diffraction (XRD) testing of the antiviral nanoparticle NM4 sample, the crystallinity, crystal type, lattice constant and phase purity of NM4 were determined. Figure 3 As shown in Figure 2, after the introduction of Mn ions into NM4, the diffraction peak position and intensity of CMM did not change significantly compared with CM, indicating that the doping of Mn ions did not have a significant effect on the crystal structure of CM. In addition, the diffraction peaks of (111), (200) and (220) crystal planes appeared in the XRD pattern of NM4, which corresponded to the standard card of metal Co (JCPDS No.15-0806), respectively. This indicates that during the calcination process, Co 2+ is reduced to metallic Co. In addition to the diffraction peaks of metallic Co, Figure 3 The diffraction peaks of (111) and (220) crystal planes of C were also observed (JCPDS No.80-0017). However, the characteristic peaks of Mn did not appear clearly, and the original diffraction peak position of Mn was shifted to the left as a whole. This shift phenomenon may be due to the local lattice distortion caused by the introduction of Mn ions. The diffraction peaks of NM4 were significantly reduced and broadened, especially the large peak near 40°, indicating that the structure of MOF after carbonization and metal oxidation was transformed into a composite with lower crystallinity, which may contain the characteristic peaks of graphitic carbon and MnO, indicating that the transformation from MOF structure to multiphase composite material was successfully achieved, which shows that NM4 material has catalytic or electrochemical activity potential.

[0028] 4. Zeta potential test was used to evaluate the charge and dispersion stability of NM4 surface, and to characterize the aggregation state and stability of NM4 particles in liquid environment. Figure 4As shown, the overall potential of the material ranges from 10.2 to 12.1 mV, indicating that the NM4 particles have moderate stability and can maintain a certain degree of dispersion in a liquid environment. The material's surface is positively charged, which favors the adsorption of negatively charged viral particles or RNA molecules, thereby promoting their binding to the target and subsequent inhibition. Furthermore, the particle size distribution of NM4 exhibits a multimodal characteristic, indicating a certain degree of heterogeneity in the material, possibly due to irregularities in the ball-and-stick interface structure or uneven element distribution. This structural feature may further affect the material's surface activity and its interaction with biomolecules.

[0029] 5. The method of identifying the functional group composition in the sample by detecting the molecular vibration absorption peak of NM4 using Fourier transform infrared spectroscopy (FTIR) technology, and analyzing the chemical structure changes, functional group introduction and intermolecular interactions on the surface of NM4. The results are as follows Figure 5 As shown, the FTIR spectrum shows a peak at about 3400 cm -1 The broad peak at 1600 cm is usually attributed to O–H or N–H stretching vibration. This absorption peak mainly comes from the water, hydroxyl groups or residual MOF structure on the surface of the material, indicating that there may be a certain degree of hydrophilic functional groups on the surface of the material or ligand residues that were not completely removed during the synthesis process, which is conducive to the adsorption of virus particles and stable dispersion. -1 The characteristic peaks on the left and right correspond to C=C aromatic structures, indicating the presence of a graphitic carbon coating in the sample, which is a conductive carbon structure formed after high-temperature treatment of the material. The peaks in the 1380-1450 cm-¹ range are usually related to C–H bending vibrations or bonds between metal ligands. This signal may be a residual trace of organic ligands in the MOF precursor, further confirming that the material is constructed through MOF derivatization. -1 The vibration peaks observed in the region are attributed to C–O stretching vibration or Mn–O vibration, indicating that the material contains certain metal oxides (such as MnO) and oxygen-containing functional groups introduced by the carbon coating structure. Finally, at 600-700 cm -1 The absorption peaks in the region are the characteristic vibrations of Co–O and Mn–O, proving that the final product contains cobalt oxide and manganese oxide components, which play an important role in the material's disease resistance and catalytic properties.

[0030] Experimental Example 2 Antiviral Ability of Nanomaterial NM4 1. Experimental sample setup The soybean plants used in the following experiments were Nandou 1138-2, a gift from Professor Li Kai of Nanjing Agricultural University and currently housed in the Plant Virology Laboratory of the College of Agriculture at Sichuan Agricultural University. Soybean seeds without apparent disease were selected for testing in an incubator at the Chengdu campus of Sichuan Agricultural University, under conditions of 25°C and a 12-hour photoperiod. The soybean mosaic virus isolate YA87 (designated SMV-YA87) was isolated and identified by this research team and deposited with the China Center for Type Culture Collection (CCTCC) on June 10, 2025, under the accession number CCTCC NO: V 202544. The deposit address is Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0031] This experiment set up 3 experimental groups, each with 3 biological replicates. The setting conditions of the experimental groups are as follows: (1) Negative control group (CK-group): no virus inoculation and no NM4 aqueous solution administration; (2) Positive control group (CK+ group): inoculated with virus but not administered with NM4 aqueous solution; (3) NM4 group: Virus was inoculated and 50 mL of NM4 aqueous solution was administered.

[0032] The specific steps are as follows: Sample plants were pre-cultured under identical environmental conditions for 10 days, then inoculated with the virus according to grouping. When normal soybean true leaves were fully expanded, an appropriate amount of quartz sand was gently sprinkled onto the leaves, ensuring a thin, even coverage. Soybean leaves infected with the soybean mosaic virus isolate YA87 and exhibiting typical symptoms (mottled leaves and wrinkled leaves) stored at -80°C were quickly transferred to 0.1 mol / L phosphate buffer (pH 7.0) and rapidly ground on ice to prepare diseased leaf puree. Next, 150 µL of the diseased sap was dripped onto the surface of a healthy soybean leaf and repeatedly rubbed with fingers until minor surface damage occurred. Finally, an additional 150 µL of diseased leaf puree was applied to ensure that healthy soybean plants were inoculated with the SMV virus. 24 hours after virus inoculation, NM4 aqueous solution was applied to the experimental group plants, and then each plant was sprayed once every 24 hours (50 mL of NM4 aqueous solution); after 7 consecutive days of spraying, samples were collected for subsequent experimental testing.

[0033] 2. Observation of plant growth characteristics like Figure 6As shown, when soybean plants with different treatments were placed together, it was observed that the soybean plants in the CK+ group inoculated with SMV virus showed obvious typical disease symptoms such as dwarfing, growth inhibition and leaf wrinkling; while the plants treated with NM4 after virus inoculation (NM4 group) had slightly better plant morphology than the CK+ group, with more normal leaf color, reduced dwarfing, and more balanced overall growth, close to the negative control group CK- group.

[0034] Observation of soybean leaves ( Figure 7 The CK- group exhibited normal green leaves, with no deformities or mosaic. The CK+ group exhibited mild mosaic symptoms, with darker leaves, some with localized yellow spots or discoloration, and variations in leaf shape, indicating viral infection. The NM4 group exhibited healthier leaves. While still somewhat affected by the virus, the overall color was more normal, and deformities and mosaic were less severe, indicating that the antiviral nanoparticle NM4 inhibited the spread of SMV to a certain extent.

[0035] The soybean plant height was measured and the results were as follows Figure 8 The data showed that the average plant height of soybeans in the CK+ group was significantly lower than that in the CK- group (p < 0.01), indicating that SMV significantly inhibited plant growth. In the NM4 group, plant height did not differ significantly from that in the CK+ group and was slightly higher than that in the CK+ group. This suggests that NM4 treatment alleviated the inhibitory effects of viral infection on soybean growth to a certain extent, demonstrating a protective effect.

[0036] 3. Transmission electron microscopy (TEM) observation of the distribution of virus particles in leaf tissue Fresh soybean leaves showing typical virus symptoms were selected from the positive control group (CK+ group) and NM4 group, and healthy control leaves were selected from the negative control group (CK- group). 1 mm 3 Small pieces of tissue were quickly pretreated in pre-chilled fixative. Samples were first fixed in 2.5% glutaraldehyde (prepared in 0.1 mol / L phosphate buffer, pH 7.2) at 4°C for 4 hours, followed by three 15-minute washes with buffer. Samples were then transferred to 1% osmium tetroxide solution and fixed at 4°C for an additional 1.5 hours. Samples were then dehydrated through a graded ethanol series (30%, 50%, 70%, 90%, and 100%), with each step lasting 18 minutes. They were then treated with 100% acetone twice, with each step lasting 0.5 hours. Following dehydration, samples were infiltrated in mixtures of epoxy resin and acetone (1:1 and 2:1, respectively) and then transferred to neat resin for infiltration overnight at room temperature. Finally, the tissue samples were added to fresh resin and polymerized at 60°C for 48 hours.

[0037] After polymerization, ultrathin sections approximately 70 nm thick were cut using an ultramicrotome and laid flat on a copper grid for later use. During the staining process, the cells were first stained with a 2% uranyl acetate solution in the dark for 15 minutes, followed by a further staining with a 3% lead citrate solution for 10 minutes. Each staining step was followed by thorough rinsing with distilled water to remove excess dye. Finally, the stained sections were observed under a transmission electron microscope (TEM) at an accelerating voltage of 80-120 kV. Areas of viral particle enrichment were identified, and the location, number, and distribution of viral particles within the cellular ultrastructure were recorded.

[0038] The results are as follows Figure 9 and Figure 10 As shown, Figure 9 The red coil in the middle shows the linear virus particles and pinwheel-shaped endosomes, and the yellow coil shows the deformed and swollen mitochondria. Figure 10 The red arrows in the middle represent the linear virus particles and the pinwheel-shaped endosomes, and the yellow arrows represent mitochondria. Figure 9 In (A)-(C), soybean mosaic virus or its inclusion bodies can be seen distributed in large quantities inside the plant, causing serious damage to the organelles. A large number of viruses are also present in the cytoplasm, and the mitochondria shrink and become smaller, and the membrane density increases. Figure 9 (D) The mitochondria in the upper right corner are normal mitochondria, and the three mitochondria in the lower left corner ( Figure 10 (A) and Figure 10 (B) Affected by the virus, the mitochondrial membrane is ruptured and the mitochondrial structure is incomplete. Figure 9 (E) shows mitochondria ruptured by surrounding viruses. Figure 9 (F) The amount of virus in the veins of the plant leaves was significantly reduced, and no obvious virus or its inclusion bodies were seen, indicating that four days after spraying the agent NM4, the amount of virus in the cells was significantly reduced compared to when it was not sprayed. Figure 9 (G)-(I), Figure 10 (C) and Figure 10 (D) It can be seen that the virus content in plant cells is significantly reduced, and there are basically no viruses in the cytoplasm; and the mitochondrial membrane is clear and the structure is intact and unchanged. Figure 10 (C) and Figure 10 (D) shows that the SMV virus content in similar mesophyll tissue parts was almost zero after application of NM4.

[0039] In summary, the virus content decreased 4 days after spraying the agent, and the virus content in the plant leaves was almost 0 7 days after spraying the agent, indicating that the nanoformulation NM4 can reduce the SMV content in soybean leaves and reduce the amount of virus accumulation.

[0040] Experimental Example 3 Analysis of plant physiological indicators The physiological indicators of soybean plants treated with the negative control group (CK- group), the positive control group (CK+ group) and the NM4 group were analyzed to explore the mechanism of action of the antiviral nanoparticle NM4.

[0041] 1. Reactive oxygen staining Using the DAB and NBT tissue staining methods, soybean leaves were soaked in 0.5 mg / mL nitro blue tetrazolium (NBT) and 1 mg / mL 3,3-diaminobenzidine (DAB) solutions, respectively, in the dark overnight. The leaves were then bleached in 95% alcohol in a boiling water bath for 15 minutes. The decolorized leaves were transferred to a paper towel saturated with 60% glycerol, and the stained soybean leaves were photographed.

[0042] The staining results are as follows Figure 11 As shown, NBT staining showed that there was no obvious blue precipitate in the leaves of the CK-group, indicating that the superoxide anion content was extremely low. A large number of blue plaques appeared in the CK+ group, indicating that the virus infection induced a large amount of superoxide anion accumulation, the oxygen concentration was high, and the cell membrane or plasma membrane was oxidized, leading to severe oxidative stress. Although blue spots were still visible in the NM4-treated group, the density and intensity were lower than those in the CK+ group, indicating that the NM4 material can reduce the level of superoxide anion accumulation induced by the virus. DAB staining showed that the leaves of the CK-group were light brown, indicating that the hydrogen peroxide content was extremely low. Obvious dark brown precipitates appeared on the leaves of the CK+ group, suggesting that the virus infection significantly induced the production of hydrogen peroxide. The staining degree of the NM4-treated group was between the CK- and CK+ groups, indicating that NM4 inhibited the virus-induced hydrogen peroxide accumulation to a certain extent.

[0043] 2. Determination of hydrogen peroxide content The specific steps are: (1) Take 0.5 g of leaves, add 2 mL of phosphate buffer (pH = 7.8) pre-cooled at 4°C, and grind into a slurry.

[0044] (2) Transfer the grinding solution to a 25 mL volumetric flask, then rinse the sample several times with phosphate buffer, combine the washing solution into the volumetric flask and dilute to the mark.

[0045] (3) After mixing evenly, place it in a refrigerator at 5°C for 10 minutes. Finally, transfer the solution to a centrifuge tube, centrifuge it at 4000 rpm for 15 minutes, and collect the supernatant.

[0046] (4) Take 10 mL of the supernatant and divide it equally into three test tubes, two of which are sample tubes and the other is a blank tube.

[0047] (5) Sodium chloride solution (0.2 mL), pH 7.8 buffer (1.5 mL), and distilled water (1.0 mL) were added to two measuring tubes and the measurement was performed using a spectrophotometer.

[0048] Figure 12 The results of hydrogen peroxide content showed that compared with the CK- group, the hydrogen peroxide content in the CK+ group was significantly increased, while the hydrogen peroxide concentration after NM4 treatment (NM4 group) was significantly lower than that in the CK+ group, approaching the normal level, indicating that the NM4 material can effectively reduce the oxidative stress caused by viral infection.

[0049] 3. Determination of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) enzyme activities The SOD, POD, and CAT activities were determined according to the method of Shang et al., 2023. The results are shown in Figure 2. Figure 13-15 shown.

[0050] As can be seen in the figure, the enzyme activities of superoxide dismutase, peroxidase, and catalase in soybean leaves in the CK+ group were significantly higher than those in the CK- group, indicating that the plants activated their antioxidant defense system after being exposed to virus stress. After the application of NM4, the activities of the three antioxidant enzymes, while still higher than those in the CK- group, decreased slightly compared to the CK+ group, indicating that NM4 treatment was effective in alleviating virus-induced oxidative stress and reducing the overactivation of defense responses.

[0051] 4. MDA content determination MDA reflects the degree of lipid peroxidation, and its content determination steps are as follows: (1) Grind 0.5 g of fresh leaves with 4 mL of 0.1% TCA (trichloroacetic acid), centrifuge at 5000 r to obtain the supernatant.

[0052] (2) Take 1 mL of supernatant and mix it with 3 mL of 0.6% TBA (thiobarbituric acid), and let it stand in a boiling water bath for 30 minutes. The blank control is 1 mL of 0.1% TCA. Finally, the colorimetry is performed at 450, 532 and 600 nm respectively.

[0053] The results of MDA content determination in soybean leaves are as follows Figure 16 As shown in the results, SMV infection significantly increased MDA levels in soybean leaves compared to the CK- group, reflecting severe oxidative stress and membrane lipid peroxidation. While the NM4 group showed a decrease in MDA levels, the difference was not significant compared to the CK+ group, indicating that NM4 did not significantly improve membrane damage.

[0054] 5. Determination of relative conductivity Take five fresh leaves from the test plant, cut them into small pieces of uniform size (approximately 0.5 cm x 0.5 cm), clean them, and place them in 10 mL of deionized water. Place them in a constant temperature shaker at 37°C for 2 hours, and then measure the initial conductivity (R1) of the solution. Then, heat the sample in a boiling water bath for 15 minutes to completely rupture the cells, and after cooling to room temperature, measure the final conductivity (R2). The formula for calculating relative conductivity is: ; Wherein, R is the relative conductivity, %; R1 is the initial conductivity, S / m; R2 is the final conductivity, S / m.

[0055] Figure 17 The results of electrical conductivity measurements of soybean leaves show that compared to the CK- group, the conductivity of the CK+ group increased, reflecting damage to the cell membrane system and increased leakage. The conductivity of the NM4 group was slightly higher than that of the CK+ group, but the difference was not significant. This is due to the positive charge of the NM4 agent itself.

[0056] 6. Chlorophyll content determination Take fresh plant leaves, wipe off the dirt on the surface of the tissue, cut into pieces (remove the midrib) and mix well. Weigh 0.2g of fresh sample × 3 parts, put it in a mortar, add 0.2g of quartz sand, 0.1g of calcium carbonate powder and 3mL of 95% ethanol to grind into a homogenous slurry, then add 10mL of ethanol and continue grinding until the tissue turns white. After standing for 5 minutes, filter, dilute the filtrate to 25mL with ethanol, and shake well. Pour the chloroplast pigment extract into a colorimetric cup, use 95% ethanol as a blank control, and measure the absorbance (A) at wavelengths of 665nm and 649nm. 665 and A 649 ), the calculation formulas of chlorophyll and total chlorophyll content are as follows: ; ; ; Wherein, a is the content of chlorophyll a, mg / g; b is the content of chlorophyll b, mg / g; c is the content of total chlorophyll, mg / g; A 663 is the absorbance of the extract at 663 nm, L / (g˖cm); A 645 is the absorbance of the extract at 645 nm, L / (g˖cm); V 总 is the total volume of the extract, mL; w is the fresh weight of the sample, g.

[0057] Figure 18The results of soybean leaf chlorophyll content measurements show that SMV treatment significantly reduced the three chlorophyll types in the CK+ group compared to the CK- group, indicating that the virus inhibits the photosynthetic system. Chlorophyll b content in the NM4 group was significantly higher than in the CK+ group (p<0.001), approaching that of the CK- group. Treatment with the chemical had no significant effect on chlorophyll a content, but showed a recovery effect on total chlorophyll content compared to CK+ treatment. This suggests that the NM4 material promotes chlorophyll b synthesis, maintains plant photosynthetic function, and mitigates the negative effects of the virus on photosynthesis.

[0058] 7. Determination of soluble protein content The soluble protein content of soybean leaves was determined using the Coomassie Brilliant Blue G-250 method. The procedure was as follows: An appropriate amount of fresh soybean leaves were homogenized in pre-chilled phosphate buffered saline (PBS, pH 7.0). The supernatant was collected as the protein extract, and Coomassie Brilliant Blue G-250 staining solution was added and mixed. After reacting at room temperature for 2 minutes, the absorbance of the mixture was measured at a wavelength of 595 nm (A). 595 The protein concentration was calculated based on the bovine serum albumin (BSA) standard curve. Figure 19 shown.

[0059] As can be seen from the figure, in terms of soluble protein content, the protein level of the NM4 group was significantly higher than that of the CK+ group and the CK- group (p<0.001), indicating that the NM4 material can induce the accumulation of resistance proteins or stress resistance proteins and enhance the overall defense ability of the plant.

[0060] Experimental Example 4 Antiviral Activity Detection In order to further study the application of nanomaterial NM4 in plant pesticide application, this experiment further subdivided the NM4 group into two main application methods: root infusion and foliar spraying: ① Root application treatment group: 50 mL of 150 μmol / L NM4 aqueous solution was slowly perfused into the roots of soybean plants (root application irrigation will cause loss); ② Foliar spraying treatment group: 20 mL of 150 μmol / L NM4 aqueous solution was sprayed on soybean leaves.

[0061] 1. RT-PCR detection of virus infection Soybean plants that had grown the first true leaf from the negative control group (CK- group), the positive control group (CK+ group) and the NM4 group (root application treatment group and foliar spray treatment group) were selected. The inoculated leaves were sampled, and the samples were quickly transferred to cryovials and quickly frozen with liquid nitrogen to maintain the integrity of their RNA. The samples were amplified by RT-PCR and then subjected to agarose gel electrophoresis. Finally, the electrophoretic bands and their positions were observed on a gel imager, and the sizes of the amplified products were compared with nucleic acid molecular weight standard markers.

[0062] Figure 20 is the SMV content in the leaves of the plants after root application. Figure 21 Comparison of foliar and root application. From left to right, the virus content after root and foliar application is compared. CK+ represents the treatment group inoculated with the virus but without NM4 aqueous solution. The results show that NM4 effectively inhibits SMV content regardless of root or foliar application, demonstrating that this nanomaterial can enter the plant through multiple pathways and improve plant disease resistance. Clear viral bands were detected in the SMV-inoculated group (CK+), indicating successful viral replication in the soybean plant. However, viral bands were significantly reduced in the NM4-treated group, indicating that the NM4 formulation effectively inhibits SMV accumulation.

[0063] 2. qRT-PCR detection of viral coat protein gene expression Total RNA was extracted from soybean leaves using the TRIzol method and transcribed into cDNA using reverse transcriptase. Subsequently, quantitative real-time PCR (qRT-PCR) was performed on a Bio-Rad iCycler to verify gene expression and determine the relative expression of SMV genes. Each treatment was replicated three times. The qRT-PCR reaction system is shown in Table 1, and the reaction procedure is shown in Table 2. Fluorescence data were collected every 0.5°C increase in temperature. After cycling, melting curves revealed that the generated products were all single species. Fluorescence values ​​of the internal reference gene β-actin were used as an internal standard. All qRT-PCR analyses used the 2-ΔΔCT method to analyze relative gene expression levels.

[0064] Table 1 qRT-PCR reaction system

[0065] Table 2 qRT-PCR reaction procedure

[0066] The V3 leaves of soybean plants in the negative control group (CK- group), the positive control group (CK+ group) and the NM4 group were sent to the company for transcriptome sequencing. If the soybean plants not inoculated with SMV (CK- group) were used as the benchmark, the relative expression level was set to 1. The sequencing results were as follows: Figure 22 As shown, after inoculation of SMV virus, the soybean plants in the CK+ group SMV CP The gene expression level increased significantly, about 14.3 times that of the uninoculated group (CK- group), indicating that SMV virus accumulated in large quantities in soybean plants. Plants after application of the drug (NM4 group) SMV CP The gene expression level dropped to about 11.5 times that of the uninoculated group, which was lower than that of the CK+ group, indicating that the agent NM4 had a certain virus inhibition effect, but failed to completely block the accumulation of viruses. The overall trend was consistent with the typical physiological response of plants to SMV infection and pesticide treatment.

[0067] like Figure 23 As shown, the volcano plot results reveal a large number of significantly differentially expressed genes (DEGs) between samples. Using a log2FoldChange > 2 and an adjusted P-value < 0.05 as screening criteria, red dots represent upregulated genes, blue dots represent downregulated genes, and gray dots represent genes with no significant changes. Overall, the number of upregulated genes in this transcriptome differential analysis was significantly higher than that of downregulated genes, indicating that a large number of defense- and metabolism-related gene expression programs were activated across the plants under the treatment conditions.

[0068] After GO enrichment analysis in the differentially expressed genes (DEGs) cluster, the results showed that ( Figure 24 ), these genes showed significant enrichment in the three categories of biological process (BP), cellular component (CC), and molecular function (MF). In the biological process (BP) category, differentially expressed genes were primarily enriched in functional terms such as fatty acid response, jasmonic acid response, photosynthesis, and light response, indicating that the plants activated biological processes related to energy metabolism and defense responses after treatment. In the cellular component (CC) category, enrichment results showed that differentially expressed genes were mainly localized to organelles and subcellular structures such as the chloroplast membrane-associated complex and plastids, indicating that plant cells underwent significant cellular structural changes under adverse or stimuli. In the molecular function (MF) category, significant enrichment in terms such as monooxygenase activity, sugar binding, and oxidoreductase activity indicated that the differentially expressed genes were involved in important biochemical functions such as reactive oxygen species regulation and metabolic transformation. Overall, the results of the GO enrichment analysis reflect that plants adapted to environmental changes under these treatment conditions through multi-level biological processes such as regulating photosynthesis, activating defense mechanisms, and remodeling cellular structure.

[0069] KEGG enrichment was performed on transcriptome genes, and the enrichment results were as follows Figure 25 shown. Figure 25 The analysis revealed significant enrichment of functions related to signal transduction, plant–pathogen interaction, and hormone signal transduction, indicating that the analyzed gene set is closely related to the plant resistance regulatory network. Furthermore, genes involved in metabolic pathways (such as protein metabolism, lipid metabolism, and amino acid metabolism) were also significantly enriched, suggesting that these metabolic changes may provide energy and metabolic intermediates to support defense responses.

[0070] From KEGG, we selected hormone-related metabolic pathways and related genes for heat map analysis. The results are shown in Table 3 and Figure 26 shown.

[0071] Table 3 Hormone pathway defense-related genes Gene name Gene function <![CDATA[log2FoldChange]]> Glyma.20G201400 Participates in the synthesis of furan fatty acids in soybeans 6.62790456 Glyma.14G217700 Auxin response factor ARF 3.48452165 Glyma.19G161000 Encodes the auxin-responsive protein IAA 3.33316986 Glyma.10G031900 Encodes an auxin-responsive protein of the SAUR family 3.23823461 Glyma.04G038100 Encodes a membrane-associated kinase regulator 2.99507203 Glyma.17G065300 Participates in cell wall remodeling 2.96113326 Glyma.08G100100 Encodes auxin response factor 2.11883366 Glyma.06G126100 Encodes abscisic acid receptor 2.04865738 Glyma.10G158000 Encoding the gibberellin receptor GID1c1 1.79832228 Glyma.06G063500 Encodes the B-type response regulator ARR18 1.63126548 Glyma.08G200700 Encoding phytochrome-related protein 1 1.55383632 Glyma.10G021500 Involved in auxin signaling 1.3449695 Glyma.08G188300 Encoding SnRK involved in energy metabolism stress response 1.17551028 Glyma.06G187000 Related to soybean column height traits 1.16119842 Glyma.02G151100 Encoding the gibberellin receptor GID1b1 1.04051652 Glyma.12G236700 Encoding CCD1 enzyme, carotenoid -1.03410578 Glyma.10G167300 Encodes GmEIN4b, an important component of the ethylene pathway -1.20486045 Glyma.13G202200 Encoding CCD1 enzyme, carotenoid -1.37221236 Glyma.15G127200 Plays a role in jasmonic acid-mediated signaling pathways -1.51271893 Glyma.13G341100 Related to membrane structure -1.58941437 Glyma.10G022900 Encodes the gibberellin receptor GID1b2 -1.60179208 Glyma.17G232255 Lipid metabolism, involved in the synthesis of unsaturated fatty acids -1.60941291 Glyma.01G204200 Belongs to the SnRK2 subfamily and is involved in ABA signaling and stress response -1.65053995 Glyma.09G040000 Participate in the mitogen signaling pathway -1.85929795 Glyma.14G162100 Encoded PP2C involved in ABA signaling pathway -1.93553978 Glyma.12G236650 Encoding CCD1 enzyme, carotenoid -1.94785486 Glyma.01G086700 Encodes MYB transcription factor -2.32050299 Glyma.17G211000 Encodes MYB transcription factor -2.32962025 Glyma.13G041800 Encodes abscisic acid receptor -2.3847472 Glyma.20G087000 Participate in the ethylene signaling pathway -2.54115583 Glyma.13G181900 Participate in plant secondary metabolism -2.56341789 Glyma.11G051800 P450 family has anti-reverse metabolic effect -3.60951719 Glyma.14G041500 Participates in the ethylene signaling pathway and affects plant resistance to viruses -3.68571588 Glyma.03G148300 Encoding the gibberellin receptor GID1b3 -5.42199088 Table 3 and Figure 26 The results showed that the action of NM4 agent involves the activation and coordination of multiple plant hormone pathways, among which the gibberellins, cytokinins, auxins and ABA signaling pathways are the most prominent. This shows that the agent NM4 can not only promote the growth and development of plants, but also enhance resistance and stress response capabilities by activating defense-related hormone signals (such as ABA, ethylene, JA).

Claims

1. A method for preparing an antiviral nanoparticle NM4, characterized in that: The following steps are involved: S1. Dissolving a cobalt salt and an organic ligand in water respectively, and then mixing them so that the molar ratio of the cobalt salt to the organic ligand in the mixture is 1.5-2.5:0.5-1.5; then reacting at 90-110° C. for 0.5-1.5 hours, and then centrifuging, washing, and drying at -35 to -30° C. for 22-26 hours to obtain Co-MOF; the organic ligand is 2,5-dihydroxyterephthalic acid, dimethylimidazole, or NN, dimethylformamide; S2. Mixing manganese salt and alkali solution to obtain solution A, dispersing Co-MOF in water to obtain solution B; then adding solution A to solution B, stirring and reacting at room temperature for 10-14 hours, centrifuging, washing, and drying at -35~-30°C for 22-26 hours to obtain CoMn-MOF; the ratio of the manganese salt, alkali solution and Co-MOF is 0.1-0.3 mmol:15-25 mL:0.3-0.5 g; S3. Place the CoMn-MOF in a protective atmosphere and heat treat it at 600-800° C. for 1-3 h to obtain the antiviral nanoparticle NM4.

2. The method for preparing the antiviral nanoparticle NM4 according to claim 1, characterized in that: The cobalt salt is cobalt acetate or cobalt chloride.

3. The preparation method of the antiviral nanoparticle NM4 according to claim 1, characterized in that: The manganese salt is manganese chloride or manganese carbonate, and the alkali solution is ammonia water or sodium hydroxide.

4. The method for preparing the antiviral nanoparticle NM4 according to claim 1, characterized in that: The centrifugal speed in S1 and S2 is 4000-5000 rpm / min, and the time is 3-7 min.

5. The method for preparing the antiviral nanoparticle NM4 according to claim 1, characterized in that: The washing liquid used in the washing in S1 and S2 is water and / or anhydrous ethanol.

6. The method for preparing the antiviral nanoparticle NM4 according to claim 1, characterized in that: The protective atmosphere is nitrogen or argon.

7. The method for preparing the antiviral nanoparticle NM4 according to claim 1, characterized in that: The heating rate in S3 is 2-5°C / min.

8. The method for preparing the antiviral nanoparticle NM4 according to claim 1, characterized in that: The heat treatment temperature is 700° C., and the heat treatment time is 3 hours.

9. An antiviral nanoparticle NM4, characterized in that: The method is prepared according to any one of claims 1 to 8.

10. Use of the antiviral nanoparticle NM4 according to claim 9 in the preparation of a preparation for treating and / or preventing soybean mosaic virus.

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