Nano-carrier, nano-pesticide preparation and preparation and application thereof

Abstract

The invention discloses a nano carrier, a nano pesticide preparation and preparation and application thereof, and belongs to the technical field of pesticides. The nano-carrier is formed by a pluronic amphiphilic polymer and a silicate ester compound through molecular self-assembly, and has a unique structure of'hydrophobic core-hydrophilic shell ', an epitaxial polyethylene glycol hydrophilic chain of the nano-carrier significantly improves the aqueous phase dispersion stability of a system, and a hydrophobic cavity efficiently encapsulates a fat-soluble pesticide through hydrophobic interaction to form a nano-preparation. The technology can effectively solve the problems of poor water solubility and low dispersity of fat-soluble pesticides, the transmembrane transport efficiency of drugs in plants is enhanced by utilizing a nano-size effect while the activity of the raw drugs is maintained, targeted delivery is realized, the use of organic solvents is remarkably reduced, the safety to non-target organisms is improved, and the application prospect is broad. And an efficient and environment-friendly novel solution is provided for preventing and treating plant diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of pesticides, and specifically relates to a nano-carrier, a nano-pesticide formulation and a preparation method thereof, and the application of the nano-pesticide formulation in the prevention and control of agricultural diseases. Background Art

[0002] Currently, the prevention and control technology of agricultural bacterial diseases still mainly relies on chemical agents, mainly including copper-zinc-based preparations (such as thiodiazole copper, thiazole zinc, etc.) and antibiotic preparations (such as kasugamycin). Although these traditional pesticides can control diseases through direct bactericidal effects, they have significant technical defects: First, the organic solvents contained in the preparations may cause environmental toxicity; second, the poor dispersibility of the dosage form leads to low utilization rate of the active ingredient; and the most crucial is that the bactericidal mechanism with a single action target is prone to cause the generation of drug resistance in pathogenic bacteria.

[0003] In response to these technical limitations, agricultural nano-pesticides developed based on nanotechnology have shown significant advantages. Nanomaterials, with their small size, large specific surface area, and targetable modification characteristics, provide a new solution for the innovation of pesticide formulation technology. Compared with traditional pesticides, nano-pesticides can not only improve the utilization rate of pesticides, reduce pesticide waste and environmental pollution; at the same time, enhance the control effect, reduce the amount of pesticides used, and reduce the impact on non-target organisms; their nano-scale dispersion system can also significantly improve the stability of pesticides. Currently, the mainstream nano-carrier systems include three categories: organic / inorganic hybrid composite materials (including metal-organic framework structures), organic polymer materials (including natural and synthetic polymer materials), and inorganic polymer materials (the typical representatives are nano-silica, graphene, and molecular sieve materials). Among them, nano-silica shows significant application value in the field of pesticide effective loading and controlled release technology due to its advantages such as high specific surface area characteristics, diverse structural forms, interface functionalization modification characteristics, and environmental degradability.

[0004] Particularly noteworthy is that nano-silica not only has excellent drug-loading performance, but its silicon element, as the second most abundant element in the earth's crust, has outstanding biocompatibility: on the one hand, it can form a silica deposition layer on the surface of the plant cell wall to enhance the physical barrier function; on the other hand, it can activate the plant's internal defense response mechanism and reduce oxidative stress damage. However, current nano-pesticides still face development bottlenecks such as immature preparation processes, high costs, and insufficient safety evaluations. These problems are more prominent in the application of lipophilic pesticides. Traditional formulations often require the addition of a large amount of organic solvents to improve their water solubility, which not only increases environmental risks but also reduces economic benefits.

[0005] Therefore, the development of a nano-carrier system with controllable physicochemical parameters to achieve efficient encapsulation and targeted delivery of lipophilic pesticides will be the key to breaking through the existing technical bottlenecks. This new delivery system is expected to simultaneously address multiple requirements such as drug stability, environmental compatibility, and plant immune activation, providing innovative solutions for the development of green agriculture. Summary of the Invention

[0006] Aiming at the problems existing in the prior art, this application provides a silica cross-linked micelles (SCLMs) nano-carrier and a nano-pesticide formulation prepared based on this, and discloses the corresponding preparation method. The Pluronic amphiphilic polymer and silicate ester form a nano-carrier with a hydrophobic cavity inside and a polyethylene glycol hydrophilic chain outside through self-assembly; the hydrophobic cavity efficiently encapsulates lipophilic pesticides through hydrophobic interaction, and the extended polyethylene glycol hydrophilic chain significantly improves the aqueous-phase dispersion stability of the system. This design not only solves the core problem of poor dispersibility of lipophilic pesticides, but also enhances the transmembrane transport efficiency of drugs in plants through the nano-size effect (10 - 70 nm), achieving the dual goals of targeted delivery and ecological friendliness.

[0007] To achieve the above object, the technical solution adopted by the present invention is as follows: A nano-pesticide formulation, comprising: a core-shell structured nano-drug delivery system formed by the self-assembly of a Pluronic amphiphilic polymer and a silicate ester compound, wherein:

[0008] The inner core is a hydrophobic cavity that encapsulates lipophilic pesticide active ingredients through hydrophobic interaction;

[0009] The hydrophilic outer shell has a polyethylene glycol hydrophilic chain segment for enhancing the aqueous-phase dispersion stability of the system.

[0010] This application provides a nano-carrier system formed by the self-assembly of a Pluronic amphiphilic polymer and a silicate ester to form a core-shell structure, comprising: a hydrophobic inner core that forms a cavity for encapsulating hydrophobic substances; a hydrophilic outer shell that contains a polyethylene glycol chain segment; the average particle size of the nano-carrier is 10 - 70 nm.

[0011] This application also provides a preparation method for the above nano-pesticide formulation, comprising the following steps:

[0012] 1) Mix the Pluronic amphiphilic polymer with an acidic solution to form a reaction medium, and optionally add an organic phase regulator;

[0013] 2) Add a silicate ester compound to the system of step 1) to form a core-shell structured nano-carrier through interfacial self-assembly;

[0014] 3) Add a silane terminator to cure the carrier structure;

[0015] 4) The SCLMs nanocarriers are obtained through dialysis and phase separation purification;

[0016] 5) After dissolving the lipophilic pesticide in an organic solvent, it is loaded onto the SCLMs core through hydrophobic interaction to prepare the nano-pesticide formulation.

[0017] Furthermore, in step 1), the organic phase regulator includes any one of cyclohexane, cycloheptane, cyclopentane, n-hexane, n-heptane, n-pentane; the molar ratio of the Pluronic amphiphilic polymer to the organic phase regulator is 1:0 to 200.

[0018] Furthermore, the molecular weight of the Pluronic amphiphilic polymer is 6000 to 20000, preferably Pluronic F108 and Pluronic F127; the acidic solution is any one of dilute hydrochloric acid, dilute sulfuric acid, dilute phosphoric acid, dilute nitric acid, citric acid, oxalic acid; the molar ratio of the Pluronic amphiphilic polymer to the acidic solution is 1:500 to 2000; the silicate compounds are selected from any one or several of tetramethyl silicate, tetraethyl silicate, tetrapropyl silicate, tetrabutyl silicate, methyltrimethoxysilane, vinyltriethoxysilane; the molar ratio of the silicate compounds to the Pluronic amphiphilic polymer is 20 to 200:1.

[0019] Furthermore, in step 3), the silane terminator is any one or several of diethoxydimethylsilane, dimethoxydimethylsilane, diisopropoxydimethylsilane, tert-butyl(methoxy)dimethylsilane, tert-butyl(hept-6-yn-1-yloxy)dimethylsilane, tert-butyldimethylhydroxyethoxysilane, bis(4-aminophenoxy)dimethylsilane, preferably diethoxydimethylsilane; the molar ratio of the silane terminator to the silicate compound is 1:1 to 100; after adding the silane terminator, it is necessary to continuously stir and react at 0 to 30 °C for 0.5 to 24 h.

[0020] Furthermore, in step 5), the organic solvent is selected from any one of N,N-dimethylformamide, dimethyl sulfoxide, dichloromethane, chloroform, tetrahydrofuran, acetonitrile, acetone, ethyl acetate.

[0021] Furthermore, the molecular weight of the lipophilic pesticide active ingredient is 100 to 800 g·mol -1 , and the mass ratio of the lipophilic pesticide to the SCLMs carrier is 1:10 to 100.

[0022] The above nano-pesticide formulation can be applied to the control of plant diseases, and the plant diseases include but are not limited to disease types such as rice bacterial blight, citrus canker, bacterial angular leaf spot, bacterial wilt, soft rot, fire blight, etc.

[0023] The beneficial effects of the present invention are:

[0024] 1. Innovatively, this application utilizes the molecular self-assembly characteristics of Pluronic amphiphilic polymers and silicate esters to construct a nano-drug delivery system with a "hydrophobic core - hydrophilic shell" structure. Through hydrophobic interactions, the hydrophobic cavity can efficiently encapsulate lipophilic pesticide active ingredients. Meanwhile, the extended hydrophilic polyethylene glycol chains significantly enhance the aqueous phase dispersion stability of the system, achieving efficient loading and stable dispersion of pesticide active ingredients and successfully solving the core technical problem of poor dispersibility of lipophilic pesticides in the aqueous phase system. On the basis of maintaining the activity of the original drug, through the nano-size effect (10 - 70 nm), the transmembrane transport efficiency of the active ingredient in plants is enhanced, realizing the efficient delivery of the drug to the target site.

[0025] 2. The nano-drug delivery system constructed in this application forms a "hydrophobic core - hydrophilic shell" structure through molecular self-assembly. The hydrophobic core is used to efficiently encapsulate lipophilic pesticide components, and the polyethylene glycol chains in the hydrophilic shell provide enhanced water interaction, enabling the lipophilic pesticide active ingredients that were originally dispersed depending on organic solvents to be directly and stably dispersed in the aqueous phase environment. The synergistic effect of this amphiphilic structure breaks through the technical bottleneck that a large amount of organic solvents must be used to maintain drug dissolution and dispersion in traditional formulations. In addition, compared with traditional pesticide formulations, the dispersion carrier of the pesticide active ingredient in this system is replaced by a nano-scale aqueous dispersion system, avoiding the environmental pollution problem caused by the loss of organic solvents with the drug after spraying. The steric hindrance effect formed by the hydrophilic chains on the surface of the nano-carrier can also effectively inhibit the aggregation and sedimentation of drug particles, further reducing the need for adding excipients such as stabilizers. This green preparation method not only reduces production costs but also meets the strict requirements of the current pesticide industry for cleaner production.

[0026] 3. After systematic toxicological verification, this preparation exhibits excellent biocompatibility: it neither affects the germination and normal growth of crop seeds nor controls the acute toxicity to non-target organisms such as zebrafish at the "slightly toxic" level, significantly reducing the environmental risk of traditional pesticides.

[0027] 4. The Bromo@SCLMs nano-drug delivery system disclosed in this application significantly enhances the crop's defense ability against oxidative stress by activating the endogenous antioxidant enzyme system of rice. This system can effectively improve the activities of superoxide dismutase and peroxidase, promote the scavenging efficiency of reactive oxygen species, and at the same time inhibit the excessive accumulation of hydrogen peroxide and lipid peroxidation reactions, thereby reducing the degree of cell membrane damage. This technology provides comprehensive oxidative stress protection for rice, not only enhancing the stress resistance of crops under adverse conditions but also improving their growth adaptability and yield stability, providing an environmentally friendly new protection solution for agricultural production.

[0028] 5. This technical solution simultaneously realizes four major innovative values: breaking through solubility limitations, enhancing drug efficacy transmission, optimizing production processes, and ensuring ecological safety. It provides a practical technical path for the large-scale production of nano-pesticides and has important industrial promotion significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is a flow chart for the preparation of nano-carrier SCLMs, nano-pesticide formulation Bromo@SCLMs, and fluorescently labeled nano-carrier RBITC-SCLMs.

[0030] Figure 2 It is the particle size distribution diagram, transmission electron microscope (TEM) image, and Zeta potential diagram of the nano-carrier SCLMs and nano-pesticide formulation Bromo@SCLMs prepared in Example 1. Among them, sub-figure a is the particle size distribution diagram of SCLMs, sub-figure b is the particle size distribution diagram of Bromo@SCLMs, sub-figure c is the TEM image of SCLMs, sub-figure d is the TEM image of Bromo@SCLMs, and sub-figure e is the Zeta potential diagram of SCLMs and Bromo@SCLMs.

[0031] Figure 3 It is a statistical chart of the dynamic change of the hydrated particle size of two materials during 7 days of soaking in deionized water. Among them, sub-figure a is the statistical chart of the hydrated particle size of SCLMs, and sub-figure b is the statistical chart of the hydrated particle size of Bromo EC.

[0032] Figure 4 The spatio-temporal distribution characteristics of RBITC-SCLMs in rice plants are shown through fluorescence imaging and quantitative analysis. Among them, sub-figure a shows the law of the fluorescence distribution of RBITC-SCLMs in root, stem, and leaf tissues changing with time at different treatment concentrations, and sub-figure b is the statistical chart of the accumulation concentration of RBITC-SCLMs in rice at different time points and different concentrations.

[0033] Figure 5 It is to compare the inhibitory effects of different formulations on Xoo through antibacterial activity tests. Among them, sub-figure a is the minimum inhibitory concentration (MIC) of Bismerthiazol (conventional drug control), Bromo (free drug), Bromo EC (emulsifiable concentrate), and Bromo@SCLMs (nano-drug delivery system); sub-figure b is the minimum bactericidal concentration (MBC) of Bromo, Bromo EC, and Bromo@SCLMs.

[0034] Figure 6The comparison results of the antibacterial effects between the nano-drug delivery system and commercial preparations. Among them, subfigure a shows the phenotypic diagram of the inhibition zone formed after treating Xoo with Bromo EC and Bromo@SCLMs; subfigure b shows the quantitative statistical analysis of the diameters of the inhibition zones of the two preparations, Bromo EC and Bromo@SCLMs.

[0035] Figure 7 The evaluation results of the effects of different concentrations of Bromo@SCLMs on the pathogenicity-related characteristics of Xoo. Among them, subfigure a quantitatively evaluates the inhibitory effect of Bromo@SCLMs on the biofilm formation of Xoo by the crystal violet staining method; subfigure b shows the statistical chart of the changes in the extracellular polysaccharide (EPS) secretion of Xoo under the treatment of different concentrations of Bromo@SCLMs; subfigure c shows the statistical chart of the changes in the production of xanthomonadin by Xoo under the treatment of different concentrations of Bromo@SCLMs.

[0036] Figure 8 The evaluation results of the preventive and protective effects of Bromo@SCLMs against rice bacterial blight. Among them, subfigure a shows the comparison of the typical diseased leaf phenotypes after treatment with different concentrations of Bromo, Bromo EC, and Bromo@SCLMs; subfigure b shows the statistical analysis results of the disease index after protection and control; subfigure c shows the comparison results of the relative control effects of each treatment group; subfigure d shows the statistical chart of the quantitative measurement results of the lesion length; subfigure e shows the statistical chart of the lesion inhibition rate.

[0037] Figure 9 The evaluation results of the therapeutic effects of Bromo@SCLMs against rice bacterial blight. Among them, subfigure a shows the comparison of the typical diseased leaf phenotypes of different treatment groups (Bromo, Bromo EC, and Bromo@SCLMs) at three concentration gradients of 1 / 2×MIC, 1×MIC, and 2×MIC; subfigure b shows the change in the disease index analyzed quantitatively; subfigure c shows the comparison results of the control effects; subfigure d shows the statistical chart of the measurement results of the lesion length; subfigure e shows the statistical chart of the lesion inhibition rate.

[0038] Figure 10 The evaluation results of the oxidative stress defense ability of Bromo@SCLMs in rice. Among them, subfigure a shows the effect of Bromo@SCLMs on the SOD enzyme activity in rice; subfigure b shows the effect of Bromo@SCLMs on the POD enzyme activity in rice; subfigure c shows the effect of Bromo@SCLMs on the accumulation level of H2O2 in rice; subfigure d shows the effect of Bromo@SCLMs on the MDA content in rice.

[0039] Figure 11For the comprehensive effects of Bromo@SCLMs at different concentrations (1 / 2×MIC, 1×MIC, 2×MIC) on the growth characteristics of rice, where subfigure a is the statistical chart of the germination rate of rice seeds; subfigure b is the statistical chart of the plant height of the plants; subfigure c is the statistical chart of the fresh weight of the plants; subfigure d is the statistical chart of the dry weight of the plants.

[0040] Figure 12 For the acute toxicity effects of different test substances on zebrafish, where subfigure a is the statistical chart of the survival rate of zebrafish treated with different concentrations of Bromo@SCLMs at different time points; subfigure b is the statistical chart of the survival rate of zebrafish treated with different concentrations of Bromo EC at different time points; subfigure c is the statistical chart of the survival rate of zebrafish treated with different concentrations of SCLMs at different time points; subfigure d is the statistical chart of the survival rate of zebrafish treated with different concentrations of Bromo at different time points. Detailed implementation manners

[0041] In order to enable those of ordinary skill in the art to better understand the technical solutions, mass spectrometry diagrams, etc. of the present invention, the technical solutions of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0042] The room temperature mentioned in the embodiments refers to 25 - 28°C; all raw materials and reagents used are commercially available products.

[0043] Example 1

[0044] This example discloses a preparation method of a nano - pesticide formulation. Specifically, after preparing the nano - carrier SCLMs, a lipophilic pesticide is encapsulated in the carrier to complete the preparation. The preparation process refers to Figure 1 , and the preparation steps are as follows:

[0045] 1) Preparation of the nano - carrier SCLMs

[0046] Weigh 0.25 g of Pluronic F108 (molecular weight is 16800, the same below) into a 20 - mL glass screw - mouth bottle, add 7.5 mL of HCl (2.0 M), ultrasonically treat for 2 min, stir at room temperature for 30 min, then add 268 μL of tetraethyl orthosilicate, continue to stir for 15 min, add 40 μL of the terminator diethoxydimethylsilane, continue to stir at room temperature for 3 h. Transfer the completed reaction solution to a 25 - KD dialysis bag and dialyze in deionized water for 24 h until all HCl is completely removed, changing the water every 3 h. After dialysis is completed, collect the material, centrifuge at 8000 rpm for 10 min, and take the supernatant as the nano - carrier SCLMs, which is stored in a 4°C refrigerator. The concentration of SCLMs is determined by the freeze - drying method.

[0047] 2) Preparation of the nano - pesticide formulation

[0048] Weigh 5 mg of bromothalonil (Bromo) and dissolve it in 100 μL of dichloromethane. After complete dissolution, prepare a stock solution with a concentration of 50 μg·μL -1 using deionized water. Dilute the SCLMs to 10 mg·mL -1 . Add the Bromo stock solution to the SCLMs solution (the mass ratio of SCLMs to Bromo is 20:1), and sonicate for 5 min to obtain the final product, denoted as Bromo@SCLMs.

[0049] Use a UV-visible spectrophotometer to measure the drug loading capacity (LC) and encapsulation efficiency (EE) of SCLMs for Bromo at a certain wavelength. The LC value is 4.86% and the EE value is 97.23%.

[0050] Related performance tests

[0051] 1. Morphological characterization of SCLMs and Bromo@SCLMs: The particle size and zeta potential of the nanomaterials are measured by dynamic light scattering. Take 100 μL of the SCLMs test sample stored in a 4°C refrigerator and transfer it to a special cuvette, then quantitatively dilute it to 1 mL with deionized water. Use a particle size analyzer to measure the particle size and zeta potential value of SCLMs, and repeat the experiment three times. Dilute the successfully prepared SCLMs to 1 mg·mL -1 . Take 10 μL and drop it onto a common carbon support film, and let it dry at room temperature. Use a transmission electron microscope (TEM) to observe the morphology and particle size of SCLMs and take pictures for recording. Measure the particle size and morphology of Bromo@SCLM according to a similar method.

[0052] The results are as Figure 2 shown. The experimental results show that the morphologies of SCLMs and Bromo@SCLM are uniform, presenting a spherical shape as a whole, and their particle sizes are comparable. After loading Bromo, the zeta potential of the nano-pesticide slightly increases and approaches electrical neutrality, indicating that the drug is successfully loaded.

[0053] 2. Stability test of the nanocarrier: Add SCLMs and Bromo EC (commercial emulsifiable concentrate, 25% bromothalonil emulsifiable concentrate, Jiangsu Tuoqiu Agrochemical Co., Ltd.) to deionized water and store them in a 4°C refrigerator. Measure the particle size of SCLMs and Bromo EC in deionized water every day, repeat the experiment three times, and measure for 7 days in total. Record the particle size changes of SCLMs and Bromo EC. Compared with the traditional commercial emulsifiable concentrate formulation, the nanocarrier system shows better stability in the 7-day hydration particle size test ( Figure 3 ). This result demonstrates the structural stability advantage of the nanocarrier in the aqueous environment.

[0054] 3. Observation of the distribution of the nanocarrier in rice

[0055] 1) Construction of fluorescent dye-labeled nanocarriers RBITC-SCLMs

[0056] Rhodamine B isothiocyanate (RBITC) is a fluorescent dye with an isothiocyanate functional group. Its excitation wavelength is 550 nm and its emission wavelength is 590 nm. It is commonly used in bioimaging and labeling. Covalent connection can be achieved by the coupling reaction between the amino group on SCLMs and the ester bond of RBITC to prepare RBITC-SCLMs.

[0057] Precisely weigh 2 mg of RBITC in the dark and dissolve it fully in 1 mL of DMF solution to prepare an RBITC stock solution (2 mg·mL -1 ). Store it in the -20 °C refrigerator protected from light. Add 2 μL of (3-aminopropyl)triethoxysilane (APES) to 18 μL of N,N-dimethylformamide (DMF) and mix well. Take 2 μL of the above mixed solution and add it to 50 μL of the RBITC stock solution (2 mg·mL -1 ). After mixing well, store it in the 4 °C refrigerator protected from light and react for 24 h to activate RBITC. Add the activated RBITC solution to 5 mL of the SCLMs stock solution (10 mg·mL -1 ), then add 100 μL of triethanolamine aqueous solution (the mass ratio of triethanolamine to water is 1:1), and stir at room temperature for 24 h. After the reaction is completed, dialyze the solution with a 25 KD dialysis bag in deionized water for 24 h to remove the unreacted reagents, changing the water every 3 h. After dialysis, collect the product, which is RBITC-SCLMs.

[0058] 2) Prepare solutions of RBITC-SCLMs at 10, 4, 2 mg·mL -1 , and mix them evenly with the same volume of hydroponic nutrient solution to prepare nutrient solutions with final concentrations of RBITC-SCLMs at 5, 2, 1 mg·mL -1 respectively. Select rice plants about 8 - 10 cm in length, immerse the roots of the rice plants in the above nutrient solutions, and rinse the roots, stems, leaves and other parts of the rice plants with deionized water at 12, 24, 48, 72, 120 h respectively, and dry the water with paper towels. Observe the fluorescence distribution of RBITC-SCLMs in the rice plants through a small animal in vivo imaging system. The experimental results show that the nanocarriers can be effectively transported to the rice stems ( Figure 4 subfigure a), and with the increase of time and the concentration of RBITC-SCLMs, the accumulation of nanocarriers in the rice plants increases ( Figure 4 subfigure b).

[0059] 4. Antibacterial activity test of nano-pesticide preparations

[0060] In this test, rice bacterial blight disease was selected as an exemplary plant disease model for technical verification. It should be noted that the technical solution disclosed in this application is not limited to a single type of plant disease.

[0061] 1) In vitro bactericidal activity determination: The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined by the microbroth dilution method.

[0062] First, 25 mg of Bromo and bismerthiazol (used as a positive control in this experiment) were separately weighed and dissolved in 500 μL of dimethyl sulfoxide (DMSO), and fully dissolved and mixed to prepare a stock solution of 50 mg·mL -1 for standby. 100 mg of Bromo EC was weighed and made up to 500 μL with deionized water, and fully mixed to prepare a stock solution with the active ingredient of Bromo being 50 mg·mL -1 for standby. Bromo@SCLMs with a loaded drug effective concentration of 50 mg·mL -1 was prepared for standby.

[0063] 100 μL of the above-mentioned solutions serially diluted with TSB liquid medium were added to a sterile 96-well plate. Subsequently, 100 μL of the target bacterial solution (1 - 2×10 8 CFU·mL -1 ) was added to each well, so that the final drug concentrations were 250, 125, 62.5, 31.25, 15.625, 7.8125, 3.90625 μg·mL -1 respectively. A negative control group was set: 100 μL of bacterial solution and 100 μL of TSB liquid medium were added to each well, and a blank control group: 200 μL of TSB liquid medium. After pipetting and mixing evenly, the 96-well plate was placed in a constant temperature incubator at 28 °C for incubation until the bacterial solution in the negative control group grew to the logarithmic growth phase. The absorbance value at OD600 was measured with an enzyme-linked immunosorbent assay (ELISA) reader, and compared with the medium without inoculated bacteria to determine the MIC of bismerthiazol, Bromo, Bromo EC, and Bromo@SCLMs against Xanthomonas oryzae pv. oryzae (Xoo). 100 μL of the bacterial solution in the sterile growth wells in the above 96-well plate was aspirated onto a TSA solid plate, spread evenly, and then placed in a constant temperature incubator at 28 °C for 48 h. The concentration corresponding to no colony growth on the solid plate was the MBC.

[0064] The experimental results are as Figure 5 shown. When the concentration of Bromo@SCLMs was 31.25 μg·mL -1 , the growth of Xoo basically stopped. The MIC value of Bromo@SCLMs against Xoo was 31.25 μg·mL -1 , while at 62.5 μg·mL -1No colony growth was observed on the corresponding TSA plate, indicating that the MBC value of Bromo@SCLMs against Xoo was 62.5 μg·mL -1 . Similarly, the MIC of Bromo EC against Xoo was 31.25 μg·mL -1 and the MBC value was 62.5 μg·mL -1 , and its antibacterial effect was comparable to that of Bromo@SCLMs. The antibacterial effect of free Bromo drug was poor, with an MIC value of 62.5 μg·mL -1 and an MBC value of 125 μg·mL -1 , which was twice the MIC and MBC values of the other two formulations. In addition, the positive control drug bismerthiazol could not inhibit the growth of Xoo at a concentration of 125 μg·mL -1 , so its MIC and MBC were both greater than 125 μg·mL -1 .

[0065] 2) The bactericidal activities of Bromo@SCLMs and Bromo EC were further compared by the inhibition zone method. First, 100 μL of the target bacterial solution (1 - 2×10 8 CFU·mL -1 ) was added to the TSA plate, and the bacterial solution was evenly spread on the plate. Then, three small holes with a diameter of 5 mm were made on the plate. Solutions of Bromo EC and Bromo@SCLMs with a concentration of 31.25 μg·mL -1 were added to each small hole, and the liquid addition volume in each small hole was 40 μL. Sterile water was used as the negative control. Each plate was placed upright in an incubator at 28 °C for 48 h, and the diameters of the inhibition zones of each group were recorded.

[0066] The experimental results are as Figure 6 shown. The illustrated results show that under the same minimum inhibitory concentration (MIC) conditions, the inhibition zone diameter formed by the nano-drug delivery system Bromo@SCLMs against Xoo was larger than that of the commercial emulsifiable concentrate formulation Bromo EC, indicating better antibacterial activity.

[0067] 3) Biofilm removal experiment: The effect of Bromo@SCLMs on the biofilm formation of Xoo was investigated using the 96-well plate method.

[0068] First, a single colony of Xoo was inoculated into the TSB medium and cultured at 28 °C and 180 rpm until the logarithmic growth phase. The bacterial solution was diluted to 1 - 2×10 8 CFU·mL -1Reserve. Prepare Bromo@SCLMs with TSB medium at concentrations of 1×MIC, 2×MIC, and 4×MIC for reserve. In a 96-well plate, add 100 μL of the prepared Xoo bacterial suspension and 100 μL of Bromo@SCLMs at different concentrations to each well in sequence, so that the final drug concentrations are 1 / 2×MIC, 1×MIC, and 2×MIC (n = 3). Blank control: Add 100 μL of bacterial solution and 100 μL of sterile TSB medium to the well. Set three biological replicates for all experimental groups. After static culture in a constant temperature incubator at 28 °C for 48 h, gently suck out the medium in the 96-well plate, and then slowly add sterile water along the well wall to wash away the excess medium, repeating the operation 3 times. After washing, add 200 μL of 0.1% crystal violet solution to each well for staining for 30 min, and then gently rinse the unbound crystal violet with deionized water, repeating the operation 3 times. Then place the 96-well plate in an oven at 37 °C to dry until there are no water droplets, add 95% ethanol to each well, dissolve the crystal violet at room temperature for 15 min, and measure the absorbance at 590 nm with an enzyme-linked immunosorbent assay reader to determine the formation of biofilm.

[0069] The experimental results are as Figure 7 shown in Subfigure a of the neutron figure. Bromo@SCLMs showed a significant concentration-dependent inhibitory effect on the formation of Xoo biofilm. At the minimum inhibitory concentration (MIC), the biofilm inhibition rate could reach 78.38%; when the drug concentration increased to 2×MIC, the inhibitory effect was further enhanced to 95.2%. This dose-effect relationship confirmed the potential application value of Bromo@SCLMs as a biofilm inhibitor.

[0070] 4) Xoo extracellular polysaccharide (EPS) inhibition test: Use the phenol-sulfuric acid method to determine the effect of Bromo@SCLMs on the EPS content produced by Xoo.

[0071] The single colony of Xoo was inoculated into TSB medium and cultured at 28 °C and 180 rpm until the logarithmic growth phase for standby. The Xoo bacterial suspension was inoculated into the TSB medium containing Bromo@SCLMs at an inoculation amount of 1% (v / v). Experimental groups with final concentrations of 1 / 2×MIC, 1×MIC, and 2×MIC were prepared respectively, and were aliquoted into 50 mL conical flasks for culture. TSB medium with the same volume and without drugs was used as a control. The conical flasks were cultured in a constant temperature shaker at 28 °C and 180 rpm for 24 h. 10 mL of the bacterial solution treated with different concentrations was taken, centrifuged at 10000 rpm for 10 min, and the supernatant was taken. Three volumes of absolute ethanol were added to the supernatant, and it was precipitated for 24 h. Then it was centrifuged at 10000 rpm for 10 min to collect the crude EPS extract, and it was dried in an oven at 60 °C until constant weight. A standard curve of glucose concentration vs. absorbance was plotted. The crude extract was dissolved in 2 mL of preheated distilled water and transferred to a 15 mL centrifuge tube. After complete dissolution, 1.5 mL of 5% (w / v) phenol solution and 5 mL of concentrated sulfuric acid (98%) were added successively. After the reaction system was thoroughly mixed, it was oscillated at room temperature for 10 min, and then placed in a 25 °C water bath for 20 min. After the reaction, the absorbance value was measured at a wavelength of 490 nm using a microplate reader, and the extracellular polysaccharide (EPS) content was calculated through the established standard curve.

[0072] The experimental results are as Figure 7 shown in subfigure b of the neutron figure. Bromo@SCLMs showed a significant dose-dependent inhibitory effect on the synthesis of extracellular polysaccharide (EPS) of Xoo. When the drug concentration gradients were 1 / 2×MIC, 1×MIC, and 2×MIC, the EPS yields decreased by 6.5%, 25.35%, and 40.86% respectively compared with the control group. This concentration-effect relationship confirmed that Bromo@SCLMs could effectively interfere with the EPS biosynthesis pathway of Xoo, and the inhibitory effect was positively correlated with the drug concentration.

[0073] 5) Pyocyanin inhibition experiment: The single colony of Xoo was inoculated into TSB medium and cultured at 28 °C and 180 rpm until the logarithmic growth phase for standby. The Xoo bacterial suspension was inoculated into the TSB medium containing Bromo@SCLMs at an inoculation amount of 1% (v / v). Experimental groups with final concentrations of 1 / 2×MIC, 1×MIC, and 2×MIC were prepared respectively, and were aliquoted into 50 mL conical flasks for culture. TSB medium with the same volume and without drugs was used as a control. The conical flasks were cultured in a constant temperature shaker at 28 °C and 180 rpm for 24 h. 10 mL of the bacterial solution treated with different concentrations was taken, centrifuged at 10000 rpm for 10 min to collect the bacterial cells, and resuspended in deionized water to 1 - 2×10 8 CFU·mL -1Reserve. Take another 10 mL of the bacterial solution, centrifuge it at 5000 rpm for 10 min, remove the supernatant, add 2 mL of methanol, extract the pigment for 10 min at room temperature in the dark, centrifuge at 10000 rpm for 10 min, take the supernatant, measure the absorbance at a wavelength of 445 nm with a microplate reader, and calculate the relative concentration of bacteriochlorophyll with methanol as the blank control.

[0074] The experimental results are as Figure 7 shown in Subfigure c of the neutron image. Bromo@SCLMs showed an obvious dose-dependent inhibitory effect on the synthesis of bacteriochlorophyll in Xoo. When the drug concentration gradients were 1 / 2×MIC, MIC, and 2×MIC, the yields of bacteriochlorophyll decreased by 12.66%, 20%, and 26.86% respectively compared with the control group. This result confirmed that Bromo@SCLMs could effectively interfere with the bacteriochlorophyll metabolic pathway of Xoo, and the inhibitory effect was positively correlated with the drug concentration.

[0075] 5. Test on the in vivo control effect of the nano-pesticide formulation

[0076] 1) The in vivo antibacterial activity of Bromo@SCLMs against Xanthomonas oryzae pv. oryzae (Xoo) was evaluated using the leaf-clipping method. Three treatment groups were set up in the experiment: Bromo@SCLMs, Bromo monomer, and Bromo EC formulation. Initial concentration solutions of 1×MIC, 2×MIC, and 4×MIC were respectively prepared and mixed with an equal volume of hydroponic nutrient solution to obtain working solutions with final concentrations of 1 / 2×MIC, 1×MIC, and 2×MIC. Deionized water treatment was used as the negative control. Each group was set with 3 biological replicates.

[0077] Select rice seedlings at the 4-leaf and 1-heart stage with uniform growth (10 plants per group), immerse their roots in the hydroponic nutrient solution containing 1 / 2×MIC, 1×MIC, and 2×MIC Bromo@SCLMs for 24 h, and set up deionized water treatment as the negative control. After pretreatment, dip a surgical scissor disinfected with 75% ethanol into the logarithmic-phase Xoo bacterial solution, cut off the leaf tissue 1.5 - 2.0 cm from the leaf tip, and immerse the cut end into the bacterial solution for 10 s to complete inoculation. When the bacterial solution was slightly dry, transfer it to a hydroponic device for cultivation at a temperature of 28°C, relative humidity of 80%, light for 16 h, and darkness for 8 h. After 14 days, count the disease incidence of the rice, record the disease index and lesion length, and calculate the control effect. The disease severity grading of rice bacterial blight is shown in Table 1. The methods for measuring the protective effects of Bromo and Bromo EC against rice bacterial blight are the same as above.

[0078] Table 1 Disease severity grading of rice bacterial blight

[0079]

[0080]

[0081]

[0082] See the results in Figure 8 The control effect of Bromo@SCLMs against rice bacterial blight showed an obvious concentration dependence. With the increase of the treatment concentration, the effects of inhibiting lesion expansion and reducing the disease index were significantly enhanced.

[0083] Under the condition of 2×MIC concentration, Bromo@SCLMs showed the best control performance. As can be seen from Figure 8 subfigure c, its control efficiency reached 67.85%, which was significantly better than that of the original Bromo drug (20.44%) and the commercial formulation Bromo EC (30.54%); compared with Bromo EC, Bromo@SCLMs had a stronger inhibitory effect on lesions. Under the condition of 2×MIC concentration, the lesion inhibition rate reached 72.07%, and the inhibitory effect was almost twice that of Bromo EC (37.65%) ( Figure 8 subfigure d). These results indicate that Bromo@SCLMs have significant advantages in the protective control of rice bacterial blight and can effectively reduce the disease severity of diseased rice.

[0084] 2) Test of the therapeutic effect of the nano-pesticide formulation

[0085] The standardized leaf-clipping inoculation method was used in the experiment. The specific operation was as follows: Dip surgical scissors disinfected with 75% ethanol into the Xoo bacterial suspension in the logarithmic phase, cut off the leaf tissue 1.5 - 2.0 cm from the leaf tip, and immerse the cut end into the bacterial solution for 10 s to complete the inoculation. Select 10 rice seedlings at the 4-leaf and 1-heart stage in each group, and perform the above operations on all rice leaves. 24 h after inoculation, immerse the roots of the rice seedlings in a hydroponic system containing Bromo@SCLMs with concentration gradients (1 / 2×MIC, 1×MIC, and 2×MIC) and continue to culture at a temperature of 28°C, relative humidity of 80%, light for 16 h, and darkness for 8 h. After 14 days, count the incidence of rice, record the disease index and lesion length, and calculate the therapeutic effect. The disease grading of rice bacterial blight is shown in Table 1. The methods for measuring the therapeutic effects of Bromo and Bromo EC against rice bacterial blight are the same as above.

[0086] See the results of the treatment experiment in Figure 9, Bromo@SCLMs showed a significant dose - effect relationship against rice bacterial blight. As the concentration of the formulation increased from 1 / 2×MIC to 2×MIC, the effects of inhibiting lesion expansion and reducing the disease index were significantly enhanced, which was similar to the results of the protection test. At the concentration of 2×MIC, Bromo@SCLMs showed the best treatment effect, with a control efficiency of 69.55%, which was significantly better than that of the original Bromo drug (40.61%) and the commercial formulation Bromo EC (55.11%). Notably, the lesion inhibition rate of Bromo@SCLMs reached 79.37%, which was 25.93 percentage points higher than that of the commercial emulsifiable concentrate formulation (53.44%). These data indicate that Bromo@SCLMs not only has a preventive and protective effect but also shows significant advantages in the disease treatment stage and can effectively control the development of the disease.

[0087] 6. Antioxidant performance test of nano - pesticide formulations

[0088] 1) Effect on SOD enzyme in rice: Weigh 0.1 g of rice leaves, add 1 mL of PBS buffer solution pre - cooled to 4°C, mechanically homogenize in a cryogenic grinder at 4°C, and then centrifuge at 10000 rpm for 10 min at 4°C to collect the supernatant. Use the Superoxide Dismutase Detection Kit (NBT method) of Beyotime Biotechnology Co., Ltd. to measure the enzyme activity.

[0089] The experimental results are as Figure 10 shown in neutron figure a. The treatment with Bromo@SCLMs significantly enhanced the SOD enzyme activity in rice. Compared with the control group treated with deionized water, the SOD content in rice plants treated with Bromo@SCLMs at MIC and 2×MIC concentrations increased by 1.3 and 1.4 times respectively. The SOD enzyme activity in rice basically recovered to the level equivalent to that of healthy group rice, and its induction effect was significantly better than that of the free Bromo drug group and the Bromo EC formulation. This result indicates that the nano - carrier SCLMs can not only enhance the bactericidal effect of Bromo but also improve the stress resistance of crops by activating the plant antioxidant defense system.

[0090] 2) Effect on POD enzyme in rice: Weigh 0.1 g of rice leaves, add 1 mL of pre - cooled extraction buffer, mechanically homogenize in a cryogenic grinder at 4°C, and then centrifuge at 8000 rpm for 10 min at 4°C. Collect the supernatant and place it on ice for later use. Use the Peroxidase (POD) Activity Detection Kit of Beijing Solarbio Science & Technology Co., Ltd. to measure the POD content in the leaf tissue.

[0091] The experimental results are as Figure 10As shown in Subfigure b, the treatment with Bromo@SCLMs significantly enhanced the POD enzyme activity in rice. Compared with the control group, the treatments at MIC and 2×MIC concentrations increased the POD activity by 1.48 times and 2.24 times respectively, and the induction effect was significantly better than that of the Bromo technical group and the Bromo EC group. This result confirmed that the Bromo@SCLMs nano - formulation can not only effectively control diseases, but also enhance the stress resistance of crops by activating the plant peroxidase defense system.

[0092] 3) Effects on the content of H2O2 in rice: Weigh 0.1 g of rice leaves, add 0.9 mL of PBS buffer solution according to the ratio of weight (g): volume (mL) = 1:9, mechanically homogenize in a cryogenic grinder at 4 °C, centrifuge at 10000 rpm for 10 min at 4 °C, and take 10% of the supernatant for testing. Use the hydrogen peroxide (H2O2) detection kit from Nanjing Jiancheng Bioengineering Institute to determine the H2O2 content in the leaf tissue.

[0093] The experimental results are as Figure 10 As shown in Subfigure c, the experimental results showed that the treatment with Bromo@SCLMs significantly reduced the H2O2 accumulation level in rice plants. Quantitative analysis showed that compared with the control group, the treatments at MIC and 2×MIC concentrations reduced the H2O2 content by 28.61% and 39.47% respectively, and the scavenging effect was significantly better than that of the Bromo technical group and the Bromo EC group. This result further confirmed that the Bromo@SCLMs nano - formulation can not only effectively inhibit pathogenic bacteria, but also reduce the oxidative damage caused by diseases by regulating the plant oxidative stress response.

[0094] 4) Effects on the content of MDA in rice: Weigh 0.1 g of rice leaves, add 1 mL of pre - cooled PBS buffer solution at 4 °C, mechanically homogenize in a cryogenic grinder at 4 °C, centrifuge at 10000 rpm for 10 min at 4 °C, and take the supernatant for testing. Use the lipid oxidation (MDA) detection kit from Beyotime Biotechnology Co., Ltd. to detect the MDA content in the leaf tissue.

[0095] The experimental results are as Figure 10 As shown in Subfigure d, the treatment with Bromo@SCLMs significantly reduced the content of malondialdehyde (MDA) in rice leaves. Quantitative analysis showed that compared with the control group, the treatments at MIC and 2×MIC concentrations decreased the MDA level by 17.89% and 35.89% respectively, and the inhibition effect was significantly better than that of the Bromo technical group and the Bromo EC group. This result confirmed that the Bromo@SCLMs nano - formulation can effectively reduce the membrane lipid peroxidation damage, indicating that the nano - carrier technology can significantly enhance the protective effect of the drug on the crop cell membrane.

[0096] 7. Biosafety test of nano - pesticide formulations

[0097] 1) Effects of nano-pesticides on the germination rate and growth of rice seeds: Bromo@SCLMs was formulated into concentrations of 1×MIC, 2×MIC, and 4×MIC, and equal volumes of hydroponic nutrient solution were added and mixed to obtain three treatment groups with final concentrations of 1 / 2×MIC, 1×MIC, and 2×MIC. The negative control group was treated with an equal amount of deionized water to ensure consistent solvent conditions.

[0098] For each group, 30 plump and round rice seeds were selected, disinfected by soaking in 75% ethanol solution for 15 s, washed 3 times with sterile water, soaked for 24 h, then soaked in Bromo@SCLMs solutions of different concentrations for 24 h. Subsequently, the seeds were transferred to an incubator at 30 °C for 48 h, and the germination rate of rice seeds in each group was recorded.

[0099] The experimental results are as Figure 11 shown in Sub-figure a. The experimental results indicate that compared with the control group, nano-pesticides at different concentrations did not have a significant effect on the germination rate of rice seeds. The germination rate of rice seeds in all treatment groups was 100%, and no germination inhibition was observed.

[0100] Rice plants with 4 leaves and 1 heart and similar growth were selected. First, their roots were soaked in Bromo@SCLMs solutions of different concentrations. After 24 h, the rice plants in each group were transferred to a hydroponic device for cultivation. After 14 days, the plant height, fresh weight, and dry weight of the rice plants in each group were measured.

[0101] The experimental results are as Figure 11 shown in Sub-figures b - d. The experimental results indicate that compared with the control group, nano-pesticides at different concentrations did not have a significant effect on the growth parameters of rice plants, including height, fresh weight, and dry weight, indicating that nano-pesticides have good biosafety for rice plants.

[0102] 2) Acute toxicity experiment of nano-pesticides on zebrafish: The "semi-static method" was used for the toxicity experiment. Four test substances, namely SCLMs, Bromo@SCLMs, Bromo monomer, and Bromo EC, were set. Among them, the treatments of Bromo@SCLMs, Bromo monomer, and Bromo EC groups were based on the effective concentration of bromothalonil monomer, and gradient concentration solutions of 2, 3, and 4 mg·mL -1 were prepared respectively. Considering that the mass ratio of the carrier to the lipophilic pesticide is 20:1 during the preparation of nano-pesticides, to maintain comparability with other groups, the actual concentrations of the SCLMs group were adjusted to 40, 60, and 80 mg·mL -1Gradient concentration. Take a 2L beaker and fill it with 1L of test solutions of different concentrations. Put 10 healthy zebrafish in each group for the experiment, and at the same time set up a blank control group without drug exposure (experimental water of equal volume). Systematically observe and record the poisoning symptoms and the number of dead individuals of the fish at four time points: 24h, 48h, 72h, and 96h.

[0103] The experimental results are as Figure 12 shown. The experimental results show that during the 96h exposure period, at a high concentration of 4mg·mL -1 , the survival rate of zebrafish in the Bromo@SCLMs treatment group was close to 100%, while the survival rates of the Bromo group and the Bromo EC group were both close to 0. It is worth noting that the blank control group (i.e., the carrier SCLMs group without drug treatment) had a survival rate greater than 80% at this time point at a high concentration of 80mg·mL -1 , verifying the reliability of the experimental system. The above data indicate that the nanocarrier SCLMs can significantly reduce the acute toxicity of Bromo, and its embedding structure may reduce the risk of direct biological exposure through a sustained-release effect.

[0104] Example 2

[0105] The difference between this example and Example 1 lies in the different preparation process of the nanocarrier SCLMs: Weigh 0.2g of Pluronic F127 (molecular weight 12600) into a 20mL glass screw-cap bottle, add 7.5mL of HCl (0.85M), stir at room temperature for 45min, then add 356μL of tetraethyl orthosilicate, continue to stir for 2h, add 37μL of the terminator diethoxydimethylsilane, and continue to stir at room temperature for 3h. Transfer the completed reaction solution to a 25KD dialysis bag and dialyze in deionized water for 24h until all HCl is completely removed, changing the water every 3h. After dialysis, collect the material, centrifuge at 8000rpm for 10min, and take the supernatant as the nanocarrier SCLMs-2, which is stored in a refrigerator at 4°C.

[0106] The particle size of the nanocarrier prepared in this example is 20 - 30nm.

[0107] Example 3

[0108] The difference between this example and Example 1 lies in the different preparation processes of the nano-carrier SCLMs: Weigh 0.25 g of Pluronic F108 into a 20 mL glass screw-cap bottle, add 7.5 mL of HCl (2.0 M), and stir at room temperature for 30 min; inject 240 μL of cyclohexane, sonicate for 30 s to form a milky homogeneous emulsion, and continue to stir at room temperature for 30 min; add 268 μL of tetraethyl orthosilicate, continuously stir for 4 h, then add 120 μL of diethoxydimethylsilane (terminator), stir at room temperature for 24 h, transfer the reaction solution to a 20 KD dialysis bag, dialyze with deionized water for 24 h, collect the dialyzed product, remove cyclohexane with a rotary evaporator, centrifuge at 5000 rpm for 10 min, and collect the supernatant, which is the SCLMs-3 nano-carrier, and store it in a refrigerator at 4 °C.

[0109] The particle size of the nano-carrier prepared in this example is 30 - 40 nm.

[0110] Example 4

[0111] The difference between this example and Example 1 lies in the different preparation processes of the nano-carrier SCLMs: Weigh 0.25 g of Pluronic F108 into a 20 mL glass screw-cap bottle, add 7.5 mL of HCl (2.0 M), and stir at room temperature for 30 min; inject 240 μL of cyclohexane, sonicate for 30 s to form a milky homogeneous emulsion, and continue to stir at room temperature for 30 min; add 348 μL of tetrapropyl orthosilicate, continuously stir for 10 h, then add 120 μL of diethoxydimethylsilane (terminator), stir at room temperature for 24 h, transfer the reaction solution to a 20 KD dialysis bag, dialyze with deionized water for 24 h, collect the dialyzed product, remove cyclohexane with a rotary evaporator, centrifuge at 5000 rpm for 10 min, and collect the supernatant, which is the SCLMs-4 nano-carrier, and store it in a refrigerator at 4 °C.

[0112] The particle size of the nano-carrier prepared in this example is 40 - 60 nm.

[0113] Example 5

[0114] The difference between this embodiment and Embodiment 1 lies in the different preparation processes of the nano-carrier SCLMs: Weigh 0.25 g of Pluronic F108 into a 20 mL glass screw-cap bottle, add 7.5 mL of HCl (2.0 M), and stir at room temperature for 30 min; inject 240 μL of cyclohexane, sonicate for 30 s to form a milky homogeneous emulsion, and continue stirring at room temperature for 30 min; add 428 μL of tetrabutyl orthosilicate, continuously stir for 10 h, then add 120 μL of diethoxydimethylsilane (terminator), stir and react at room temperature for 24 h, transfer the reaction solution to a 20 KD dialysis bag, dialyze with deionized water for 24 h, collect the dialyzed product, remove cyclohexane with a rotary evaporator, centrifuge at 5000 rpm for 10 min, and collect the supernatant, which is the SCLMs-5 nano-carrier, and store it in a refrigerator at 4 °C.

[0115] The particle size of the nano-carrier prepared in this embodiment is 40 - 60 nm.

[0116] The test results show that after the nano-carriers SCLMs2 - 5 prepared by the processes of Embodiments 2 - 5 (including the cyclohexane template method) are loaded with lipophilic pesticides, the prepared nano-pesticide formulations are comparable to the formulations prepared in Embodiment 1 (without cyclohexane) in terms of key performance indicators. Both show excellent plant disease control capabilities, can significantly improve the transport efficiency of pesticides in plants, and at the same time reduce the potential risks to non-target organisms. Despite the differences in the preparation processes, the functional performances of the final products tend to be consistent, indicating that different process routes can achieve the expected design goals of nano-pesticide formulations.

[0117] Embodiment 6

[0118] The difference between this embodiment and Embodiment 1 lies in the different lipophilic pesticides encapsulated in the nano-carrier SCLMs when preparing the nano-pesticide formulation: Weigh 5 mg of thifluzamide and dissolve it in 100 μL of dichloromethane to prepare a pesticide stock solution of 50 μg·μL -1 . Dilute SCLMs with deionized water to 10 mg·mL -1 , add the thifluzamide stock solution to the SCLMs solution (the mass ratio of SCLMs to thifluzamide is 20:1), and sonicate for 5 min to prepare the final product.

[0119] The above shows and describes the basic principles, main features, and advantages of the present invention. However, the above are only specific embodiments of the present invention, and the technical features of the present invention are not limited thereto. Any other embodiments obtained by those skilled in the art without departing from the technical solution of the present invention should be covered within the scope of the present invention.

Claims

1. A nano-pesticide formulation, characterized in that, Comprising: A core-shell structured nano-drug delivery system formed by the molecular self-assembly of Pluronic amphiphilic polymers and silicate compounds, wherein: The inner core is a hydrophobic cavity that encapsulates lipophilic pesticide active ingredients through hydrophobic interactions; The hydrophilic outer shell has a polyethylene glycol hydrophilic segment for enhancing the aqueous phase dispersion stability of the system.

2. A nanocarrier, characterized in that, A core-shell structure is formed by the molecular self-assembly of Pluronic amphiphilic polymers and silicate, including: A hydrophobic inner core that forms a cavity for encapsulating hydrophobic substances; A hydrophilic outer shell containing a polyethylene glycol segment; The average particle size of the nano-carrier is 10 - 70 nm.

3. The preparation method of the nano-pesticide formulation according to claim 1, wherein, Including the following steps: 1) Mix the Pluronic amphiphilic polymer with an acidic solution to form a reaction medium, and optionally add an organic phase regulator; 2) Add a silicate compound to the system of step 1) to form a core-shell structured nano-carrier through interfacial self-assembly; 3) Add a silane terminator to cure the carrier structure; 4) Purify through dialysis and phase separation to obtain the SCLMs nano-carrier; 5) Dissolve the lipophilic pesticide in an organic solvent and then load it onto the SCLMs inner core through hydrophobic interaction to prepare the nano-pesticide formulation.

4. The preparation method of the nano-pesticide preparation according to claim 3, characterized in that, In step 1), the organic phase regulator includes any one of cyclohexane, cycloheptane, cyclopentane, n-hexane, n-heptane, n-pentane; the molar ratio of the Pluronic amphiphilic polymer to the organic phase regulator is 1:0 - 200.

5. The preparation method of the nano-pesticide preparation according to claim 3, characterized in that, The molecular weight of the Pluronic amphiphilic polymer is 6000 - 20000; The acidic solution is any one of dilute hydrochloric acid, dilute sulfuric acid, dilute phosphoric acid, dilute nitric acid, citric acid, oxalic acid; The molar ratio of the Pluronic amphiphilic polymer to the acidic solution is 1:500 - 2000; The silicate compounds are selected from any one or several of tetramethyl silicate, tetraethyl silicate, tetrapropyl silicate, tetrabutyl silicate, methyltrimethoxysilane, vinyltriethoxysilane; The molar ratio of the silicate compounds to the Pluronic amphiphilic polymer is 20 - 200:

1.

6. The preparation method of the nano-pesticide preparation according to claim 3, characterized in that, In step 3), the silane terminator is any one or several of diethoxydimethylsilane, dimethoxydimethylsilane, diisopropoxydimethylsilane, tert-butyl(methoxy)dimethylsilane, tert-butyl(hept-6-yn-1-yloxy)dimethylsilane, tert-butyldimethylhydroxyethoxysilane, bis(4-aminophenoxy)dimethylsilane; the molar ratio of the silane terminator to the silicate compound is 1:1 - 100; after adding the silane terminator, it is necessary to continuously stir and react at 0 - 30 °C for 0.5 - 24 h.

7. The preparation method of the nano-pesticide preparation according to claim 3, characterized in that, In step 5), the organic solvent is selected from any one of N,N-dimethylformamide, dimethyl sulfoxide, dichloromethane, chloroform, tetrahydrofuran, acetonitrile, acetone, ethyl acetate.

8. The preparation method of the nano-pesticide preparation according to claim 3, characterized in that, The molecular weight of the fat-soluble pesticide active ingredient is 100 to 800 g·mol -1 , and the mass ratio of the fat-soluble pesticide to the SCLMs carrier is 1:10 to 100.

9. Use of the nano-pesticide formulation according to claim 1 in controlling plant diseases.

10. The application according to claim 9, characterized in that, The plant diseases include rice bacterial blight, citrus canker, bacterial angular leaf spot, bacterial wilt, soft rot, fire blight.

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