Method for treating glyphosate potassium salt resistant weeds

Abstract

The invention relates to the field of agricultural chemistry, and discloses a method for treating glyphosate potassium salt resistant weeds, which comprises the following steps: preparing a composite composition containing nanocrystallized glyphosate potassium salt, a resistance reversal agent and a delivery carrier; embedding the composite composition through a microfluidic technology to form core-shell structure nanoparticles; performing surface modification on the nanoparticles to construct environmental response characteristics; adding a bionic penetrating peptide into the modified nanoparticles, and carrying out freeze-drying stabilization; controlling the staged release of each component in the nanoparticles based on environmental parameters; and performing accurate spraying application according to field conditions. By introducing a pH / ROS dual-response mechanism, a core-shell structure constructed by microfluidic and bionic penetrating peptide modification, precise delivery and synergistic release of components are realized, and targeting, timeliness and preparation stability of resistant weed control are remarkably improved.

Description

Technical Field

[0001] The present invention relates to the field of agrochemical technology, and particularly to a method for controlling glyphosate-potassium salt resistant weeds. Background Art

[0002] With the wide application of glyphosate-potassium salt, some weed populations have gradually developed resistance, resulting in a significant decline in the efficacy of traditional herbicides. Currently, the control of glyphosate-potassium salt resistant weeds has become a major problem in global agricultural production.

[0003] In the existing field of resistant weed control, known techniques mostly adopt a multi-component compounding strategy to attempt to enhance the herbicidal effect. For example, glyphosate is co-loaded with functional components such as RNA interference factors and epigenetic regulators in a nanocarrier. However, most of these techniques use physical co-loading or simple embedding methods, and the carrier structure is often non-stratified, and the response mechanism is also non-selective. The release of the preparation mainly depends on passive diffusion, lacking the ability to sense specific environmental signals of the target tissue, resulting in low precision in controlling the release time and space of the components, and limited efficiency in complex plant tissues.

[0004] However, there are still some deficiencies in the existing technology. On the one hand, traditional nano-formulations often use physical mixing or disordered encapsulation methods, resulting in mutual interference during the release process of functional components, different release rates, and inability to respond and regulate according to the specific microenvironment in resistant weeds, thus causing imbalance in drug release and making it difficult to form effective synergistic intervention. On the other hand, the plant cuticle and cell wall pose a natural barrier to drug transport, especially with poor permeability to nucleic acid molecules and macromolecular regulators. Existing formulations generally lack a structural design with targeted penetration ability, reducing the drug efficacy due to retention or leakage. In addition, most carriers are prone to particle size growth, aggregation, and inactivation of active ingredients during storage and reconstitution, seriously affecting their field applicability and stability. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for controlling glyphosate-potassium salt resistant weeds, which solves the problems of low herbicidal efficiency, poor component synergistic effect, weak targeted release control, and insufficient preparation stability existing in the existing technology for controlling resistant weeds.

[0006] To achieve the above purpose, the present invention is realized through the following technical solutions: A method for controlling glyphosate-potassium salt resistant weeds, comprising the following steps: S1. Prepare a composite composition containing nano-sized glyphosate-potassium salt, a resistance reversal agent, and a delivery carrier; S2. Embed the composite composition by microfluidic technology to form core-shell structured nanoparticles; S3. Perform surface modification on the nanoparticles to construct environmental response characteristics; S4. Add a biomimetic penetration peptide to the modified nanoparticles and perform freeze-drying stabilization; S5. Control the staged release of each component in the nanoparticles based on environmental parameters; S6. Perform precise spraying application according to field conditions.

[0007] Preferably, the preparation of the composite composition includes: Ball-mill 280 - 320 parts by weight of potassium glyphosate with zirconium beads at a mass ratio of 5:1 - 7:1 for 4 - 6 hours to obtain nanocrystalline potassium glyphosate with a D50 of 70 - 90 nm; Mix 45 - 55 parts by weight of propyl 4-chlorophenoxyacetate with 7 - 13 parts by weight of polyacrylic acid to form an oil phase; Add 18 - 22 parts by weight of dsRNA and 8 - 12 parts by weight of RG108 to a ZIF-8 precursor solution composed of 0.2 - 0.3 mol / L Zn(NO3)2 and 0.8 - 1.2 mol / L 2-methylimidazole, and react at room temperature for 2 - 4 hours to form ZIF-8 nanoparticles loaded with dsRNA and RG108; Mix the nano potassium glyphosate, the oil phase, and the ZIF-8 nanoparticles to obtain a composite composition containing nano potassium glyphosate, a resistance reversal agent, and a delivery carrier; Among them, a resistance reversal agent such as propyl 4-chlorophenoxyacetate can induce metabolic interference in the glyphosate resistance pathway, and dsRNA is embedded in the metal-organic framework (ZIF-8) to form a stable carrier structure, which helps to delay degradation and release efficiently in target plants. Through this composite design, the chemical action of potassium glyphosate, the metabolic inhibition mechanism, and the gene silencing strategy are integrated into a carrier system, significantly enhancing the complexity and synergy of the overall governance system.

[0008] Preferably, the microfluidic technology embedding includes: Use a three-channel chip with a channel width of 180 - 220 μm to process the composite composition at a flow rate ratio of internal phase:oil phase:external phase = 1:3 - 3.5:4.5 - 5.5; Among them, the internal phase is a potassium glyphosate solution containing ethanol / water with a volume ratio of 7:3, and the oil phase contains 80 - 120 parts by weight of PLGA; The external phase contains 0.2 - 0.3 mol / L Zn(NO3)2, and react for 4 - 6 h to form core-shell structured particles; Preferably, the microfluidic technology embedding further includes: Emulsify at an oil phase temperature of 35 - 40 °C for 8 - 12 min; Collect the particles with a centrifugal force of 8000 - 10000 × g; Wash three times with 200 - 300 parts by weight of ethanol to remove unreacted substances.

[0009] The use of the microfluidic chip enables the stable formation of the internal and external phase interfaces under precise control, which helps to control the nanoparticle size and drug release behavior. The biodegradable material (such as PLGA) contained in the oil phase forms a structurally stable protective shell, and the glyphosate potassium salt and regulatory components can be simultaneously loaded inside the shell to form a core-shell spatial structure. The core-shell structure realizes the sequential delivery control by restricting the diffusion rates of different active components during the release process, and also provides a stable support for subsequent responsive modification.

[0010] Preferably, the surface modification includes: Co-incubate 20 - 30 parts by weight of DSPE-PEG2000 with the nanoparticles at 42 - 48 °C for 25 - 35 min; Graft 30 - 40 parts by weight of dimethylaminoethyl methacrylate under ultraviolet light with a wavelength of 360 - 370 nm and an intensity of 8 - 12 mW / cm 2 ; Add 20 - 30 parts by weight of lipoic acid-PEG2000 derivative to construct a ROS-responsive crosslinking network.

[0011] Preferably, the step of grafting under ultraviolet light includes: Control the reaction time for 20 - 30 min to make the polymer swelling ratio reach 2.8 - 3.2; Complete the lipoic acid crosslinking reaction in pH 7.4 PBS buffer for 2 - 4 h.

[0012] The polymer used, such as dimethylaminoethyl methacrylate, has a cracking property sensitive to pH changes, and the ROS-responsive network formed by the lipoic acid derivative can perceive the oxidation signal and activate the structural depolymerization.

[0013] Preferably, the lyophilization stabilization includes: Couple 5 - 7 parts by weight of CPP-EC1 biomimetic penetrating peptide to the nanoparticle surface through a thioether bond; Add 30 - 40 parts by weight of trehalose as a lyoprotectant; Perform three-stage lyophilization: pre-freeze at -45 - -40 °C for 4 - 6 h, primary drying at -35 °C / 100 mbar for 8 - 10 h, and secondary drying at 25 °C / 10 mbar for 4 - 6 h.

[0014] Preferably: The three-stage lyophilization includes: Maintain the shelf temperature difference of ±1.5 °C during the pre-freezing stage; The cold trap temperature in the primary drying stage is ≤ -55 °C; The residual moisture after secondary drying is ≤ 1.5%.

[0015] The conjugation of penetration peptides enhances the penetration efficiency of the formulation in the plant epidermis or cell wall, while the introduction of lyoprotectants such as trehalose ensures the structural integrity of the formulation morphology and the persistent retention of component functions.

[0016] Preferably, the staged release control includes: Trigger the release of 280 - 320 parts by weight of potassium glyphosate when pH > 7.2; Release 30 - 50 parts by weight of S-methylisothiourea sulfate under the condition that the GST enzyme concentration > 0.4 U / mL; Sustainably release 18 - 22 parts by weight of dsRNA@ZIF-8 when the H2O2 concentration > 50 μM; When the pH of the resistant weed roots increases, trigger the release of the outer layer of potassium glyphosate; when the oxidation signal is enhanced or the GST enzyme level increases, initiate the release of the gene silencing module and the metabolic reverser.

[0017] Preferably, the precise spraying application includes: Spray 0.1 mM Ca 2+ solution 200 - 300 L / ha 24 hours before spraying; Control the droplet size VMD = 150 - 200 μm, the spraying pressure 3 - 5 bar, and the traveling speed 8 - 10 km / h; Add 0.3 - 0.5% methylated vegetable oil when the environmental humidity < 60%.

[0018] By means such as preset spraying windows, regulating stomatal permeability and droplet parameters, etc., the delivery efficiency and bioaccessibility of the aforementioned formulation are systematically ensured. For example, before spraying, Ca 2+ spraying is used to induce stomatal opening, and measures such as adding adjuvants regulated by humidity changes are taken to maximize the contact efficiency of the formulation and adapt to environmental changes.

[0019] In summary, the present invention includes at least one of the following beneficial technical effects: 1. By integrating the pH and ROS dual-responsive mechanisms, the present invention realizes the precise release of functional components in the microenvironment of plants. This intelligent response system can dynamically adjust the drug release rhythm according to the acid-base conditions and oxidation states of the target tissues, effectively avoiding problems such as "fast first and then weak" or "release out of control" of conventional formulations, providing a multi-stage intervention path for resistant weeds, and reflecting the unique advantages of the present invention in temporal regulation and environmental perception.

[0020] 2. The present invention constructs a core-shell nanostructure using microfluidic technology, enabling different components to be stably encapsulated in independent layers and avoiding mutual interference between components. This structured carrier not only enhances the physicochemical stability of the preparation but also provides a good physical basis for the co-release of multiple components, demonstrating the integration ability in complex pharmacodynamic combinations and serving as an important support for the implementation of multi-target and multi-pathway intervention strategies.

[0021] 3. The present invention introduces a strategy of modifying with biomimetic penetrating peptides, significantly enhancing the penetration efficiency of nanoparticles between the plant cuticle and cell wall. The surface functionalization of the penetrating peptides endows the preparation with good tissue affinity and transmembrane ability, enabling the active ingredients to enter the target site more quickly and deeply, solving the limitation of the plant barrier on the release of drug efficacy from the source, and reflecting the technological innovation in targeted delivery and enhanced penetration.

[0022] 4. The design of the present invention integrates epigenetic regulators and RNA interference elements to construct a co-intervention system targeting the glyphosate resistance regulatory pathway. By doubly inhibiting the expression and stability of resistant genes, this combined mechanism effectively enhances the ability to physiologically interfere with resistant plants, avoiding the problem that traditional single herbicides are circumvented by the resistance pathway, and forming a highly targeted molecular-level attack strategy.

[0023] 5. The present invention improves the long-term storage performance of the preparation by introducing a freeze-drying protectant, significantly reducing the risk of particle size change and activity loss during storage and transportation. The protectant stabilizes the particle interface during drying and rehydration processes, maintaining the structural integrity and dispersion state of the preparation, providing a key guarantee for achieving stable and efficient drug efficacy in agricultural application, and enhancing the practicality and industrial transformation potential of the preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a schematic diagram of the method flow of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0025] The following will further elaborate on the present invention in conjunction with the attached Figure 1 , for a more detailed description of the present invention.

[0026] Unless otherwise specified, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods.

[0027] As Figure 1 shown: Example 1: S1. Preparation of the composite composition: 300 parts by weight of potassium glyphosate and zirconium beads are ball-milled in a ball mill at a mass ratio of 6:1 for 5 hours to obtain nano-potassium glyphosate with a D50 of 80 nm; Take 50 parts by weight of propyl 4 - chlorophenoxyacetate and mix it evenly with 10 parts by weight of polyacrylic acid as the oil phase; Prepare a solution of 0.25 mol / L Zn(NO3)2 and 1.0 mol / L 2 - methylimidazole, add 20 parts by weight of dsRNA and 10 parts by weight of RG108, and react at room temperature for 3 hours to form ZIF - 8 particles loaded with dsRNA and RG108; Mix the obtained nano - glyphosate potassium salt, the oil phase and the ZIF - 8 particles to form a composite composition.

[0028] S2, Microfluidic technology embedding: Use a three - channel chip with a channel width of 200 μm, and control the flow rate ratio of the inner phase (ethanol / water = 7:3 volume ratio), the oil phase (containing 100 parts by weight of PLGA) and the outer phase (0.25 mol / L Zn(NO3)2) to be 1:3.25:5; React under this condition for 5 hours to form nanoparticles with a uniform particle size and a stable core - shell structure.

[0029] S3, Nanoparticle surface modification: Incubate 25 parts by weight of DSPE - PEG2000 with the nanoparticles at 45 °C for 30 minutes; Subsequently, under ultraviolet light with a wavelength of 365 nm and an intensity of 10 mW / cm 2 Graft 35 parts by weight of dimethylaminoethyl methacrylate; then introduce 25 parts by weight of lipoic acid - PEG2000 derivative to construct a ROS - responsive cross - linked network to complete the surface modification.

[0030] S4, Bionic penetration and freeze - drying treatment: Couple 6 parts by weight of CPP - EC1 bionic penetration peptide to the particle surface through a thioether bond; Add 35 parts by weight of trehalose as a freeze - drying protectant; Perform a three - stage freeze - drying process: pre - freeze at - 42 °C for 5 hours, primary drying at - 35 °C / 100 mbar for 9 hours, and secondary drying at 25 °C / 10 mbar for 5 hours to obtain stable powder - state nanoparticles.

[0031] S5, Phased response release mechanism: When the pH value rises above 7.2, trigger the release of 300 parts by weight of glyphosate potassium salt; When the GST enzyme concentration exceeds 0.4 U / mL, release 40 parts by weight of S - methylisothiourea sulfate; When the environmental H2O2 concentration is higher than 50 μM, initiate the slow release of dsRNA@ZIF - 8.

[0032] S6, Precise pesticide application: Spray 0.1mMCa 24 hours before application 2+ Solution 250L / ha; The droplet size VMD is controlled to be 175μm, the spray pressure is 4bar, and the operating speed is 9km / h; When the ambient humidity is lower than 60%, add 0.4% methylated vegetable oil adjuvant to improve the spraying effect.

[0033] Embodiment 2: S1. Preparation of composite composition: 280 parts by weight of potassium glyphosate and zirconium beads were ball-milled at a mass ratio of 7:1 for 4 hours to obtain nanoparticles with a D50 of 90 nm; 45 parts by weight of 4-chlorophenoxyacetic acid propyl were mixed with 7 parts by weight of polyacrylic acid to prepare an oil phase; Prepare 0.2 mol / L Zn(NO3)2 and 0.8 mol / L 2-methylimidazole precursor solution, add 18 parts by weight of dsRNA and 8 parts by weight of RG108, and react at room temperature for 2 hours; The resulting three components are mixed to form a composite composition.

[0034] S2. Microfluidic technology embedding: A three-channel chip with a channel width of 180 μm was used, and the flow rate ratio of the inner phase: oil phase: outer phase was 1:3:4.5; The oil phase contained 80 parts by weight of PLGA, the external phase concentration was 0.2 mol / L Zn(NO3)2, and the reaction time was 4 hours.

[0035] S3. Surface modification of nanoparticles: 20 parts by weight of DSPE-PEG2000 were incubated with the particles at 42°C for 25 minutes; UV grafting uses a wavelength of 360nm and an intensity of 8mW / cm 2 The light source is grafted with 30 parts by weight of dimethylaminoethyl methacrylate; and 20 parts by weight of lipoic acid-PEG2000 derivative is added to construct a ROS response layer.

[0036] S4, Bionic penetration and freeze-drying treatment: Coupling 5 parts by weight of CPP-EC1 to the particle surface; Add 30 parts by weight of trehalose protective agent; A three-stage freeze-drying process was implemented: pre-freezing at -45°C for 4 hours, primary drying at -35°C / 100mbar for 8 hours, and secondary drying at 25°C / 10mbar for 4 hours.

[0037] S5. Phased response release mechanism: When pH>7.2, 280 parts by weight of glyphosate potassium salt is released; The GST enzyme concentration > 0.4 U / mL releases 30 parts by weight of S-methylisothiourea sulfate; The H2O2 concentration > 50 μM slowly releases 18 parts by weight of dsRNA@ZIF-8.

[0038] S6. Precise application of pesticides: Spray 200 L / ha of 0.1 mM Ca 2+ solution before pesticide application; Control the volume median diameter (VMD) of the droplets to 150 μm, the spray pressure to 3 bar, and the operation speed to 8 km / h; When the humidity < 60%, add 0.3% methylated vegetable oil as an adjuvant.

[0039] Example 3: S1. Preparation of the composite composition: Mill 320 parts by weight of potassium glyphosate with zirconium beads at a mass ratio of 5:1 for 6 hours to obtain nanoparticles with a D50 of 70 nm; mix 55 parts by weight of propyl 4-chlorophenoxyacetate with 13 parts by weight of polyacrylic acid to obtain an oil phase; Prepare 0.3 mol / L Zn(NO3)2 and 1.2 mol / L 2-methylimidazole precursor solutions, add 22 parts by weight of dsRNA and 12 parts by weight of RG108, and react for 4 hours; Mix the three evenly to form a composite composition.

[0040] S2. Embedding by microfluidic technology: Use a three-channel chip with a channel width of 220 μm, and set the flow rate ratio of the inner phase: oil phase: outer phase to 1:3.5:5.5; The oil phase contains 120 parts by weight of PLGA, the outer phase Zn(NO3)2 concentration is 0.3 mol / L, and the reaction is carried out for 6 hours.

[0041] S3. Surface modification of nanoparticles: Add 30 parts by weight of DSPE-PEG2000 and co-incubate at 48 °C for 35 minutes; Use an ultraviolet light source with a wavelength of 370 nm and an intensity of 12 mW / cm 2 to graft 40 parts by weight of dimethylaminoethyl methacrylate; add 30 parts by weight of lipoic acid-PEG2000 derivative to construct a responsive network.

[0042] S4. Bionic penetration and freeze-drying treatment: Couple 7 parts by weight of CPP-EC1 to the particle surface; Use 40 parts by weight of trehalose for freeze-drying protection; Freeze-drying process: Pre-freeze at -40 °C for 6 hours, primary drying at -35 °C / 100 mbar for 10 hours, and secondary drying at 25 °C / 10 mbar for 6 hours.

[0043] S5, Step - by - step Response Release Mechanism: Release 320 parts by weight of potassium glyphosate in an environment with pH > 7.2; Release 50 parts by weight of S - methylisothiourea sulfate when the GST enzyme concentration is higher than 0.4 U / mL; Sustainably release 22 parts by weight of dsRNA@ZIF - 8 when the H2O2 concentration is greater than 50 μM.

[0044] S6, Precision Spraying: Spray 0.1 mM Ca 2+ solution at 300 L / ha 24 hours before spraying; Control the droplet size to be 200 μm, the spraying pressure to be 5 bar, and the operation speed to be 10 km / h; When the environmental humidity is lower than 60%, add 0.5% methylated vegetable oil.

[0045] Comparative Example 1: Compared with Example 1, the difference is that dsRNA and RG108 are not added to the composite composition, that is, the gene interference module and epigenetic regulator are omitted, and only nano - potassium glyphosate and resistance reverser are included, and the rest are the same.

[0046] Comparative Example 2: Compared with Example 1, the difference is that the ball - milled particle size of potassium glyphosate is controlled at D50 = 120 nm, and the rest are the same.

[0047] Comparative Example 3: Compared with Example 1, the difference is that in the microfluidic embedding process, the core - shell structure is not used for construction, and the traditional double - emulsion method is used to prepare nanoparticles, and the rest are the same.

[0048] Comparative Example 4: Compared with Example 1, the difference is that in the surface modification process, dimethylaminoethyl methacrylate is not grafted, and the construction of the pH - responsive functional layer is omitted, and the rest are the same.

[0049] Comparative Example 5: Compared with Example 1, the difference is that in the surface modification, lipoic acid - PEG2000 derivative is not introduced, and the construction of the ROS - responsive cross - linked network is omitted, and the rest are the same.

[0050] Comparative Example 6: Compared with Example 1, the difference is that the biomimetic penetration peptide CPP - EC1 is not conjugated to the surface of the nanoparticles, and its function of enhancing the trans - cellular barrier is cancelled, and the rest are the same.

[0051] Comparative Example 7: Compared with Example 1, the difference is that in the freeze - drying process, trehalose protectant is not added, and it is changed to unprotected freeze - drying treatment, and the rest are the same.

[0052] Comparative Example 8: Compared with Example 1, the difference lies in that only the pH-responsive release mechanism of potassium glyphosate is retained in the triple-release mechanism, and the GST enzyme-responsive and H2O2-responsive modules are omitted, and the rest are the same.

[0053] Comparative Example 9: Compared with Example 1, the difference lies in that no Ca 2+ inducer and methylated vegetable oil adjuvant were added during the precise spraying stage, and the environmental adaptation adjustment strategy was cancelled, and the rest were the same.

[0054] Test Experiment 1: Experimental samples: the formulation of Example 1, the formulations of Comparative Examples 2, 3, 4, 5, and 8; Experimental steps: Particle size stability test: Weigh 10 mg of each sample and disperse it in 10 mL of pH 7.2 PBS buffer (at room temperature).

[0055] Use ultrasonic treatment for 1 min to eliminate agglomeration.

[0056] Measure the initial particle size and PDI value using a dynamic light scattering instrument (DLS).

[0057] After standing for 24 hours, measure the particle size and PDI again to evaluate the change in stability.

[0058] Encapsulation efficiency and drug loading efficiency test: Take 10 mg of the formulations of Example 1 and Comparative Examples respectively, dissolve them in 1 mL of acetic acid buffer (pH 5.0), and shake to lyse the particle structure.

[0059] After centrifuging to remove the residual carrier, collect the supernatant.

[0060] Use an ultraviolet-visible spectrophotometer to detect the contents of potassium glyphosate and RG108 therein (measure at 205 nm and 288 nm respectively).

[0061] At the same time, detect the free drug (unencapsulated part), and calculate the encapsulation efficiency (EE%) and drug loading (DL%).

[0062] Response release performance test: Prepare a release environment of pH 7.2 + 50 μM H2O2 (ROS simulation environment).

[0063] Take an equal amount of the formulation sample (10 mg) and add it to each group of solutions (10 mL), and oscillate at a constant speed in a shaking incubator at 37 °C.

[0064] Take 1 mL of sample every 2 hours and supplement it with the same volume of fresh buffer for 24 hours.

[0065] Analyze the release amounts of potassium glyphosate, RG108, and dsRNA, plot the cumulative release curves, and the experimental results are shown in Table 1.

[0066] Table 1: Comparative experimental data of particle size, encapsulation efficiency, and release performance It can be seen from Table 1 that: The core-shell nanoparticles constructed by using a microfluidic chip in the examples showed significant particle size uniformity and structural stability. In the comparative examples where this structure was removed or the particle size deviated from a specific range, the particle size increased rapidly and the PDI increased, indicating an obvious aggregation trend of the preparation in the aqueous environment. This difference stems from the fluid shear and interface stability control mechanisms achieved by microfluidic technology, which effectively ensured the particle encapsulation efficiency and physical uniformity, providing a stable carrier basis for subsequent targeted release.

[0067] Glyphosate was rapidly released under alkaline conditions, while RG108 and dsRNA showed highly responsive release under ROS conditions. For the samples in the control group lacking a pH-responsive layer, ROS cross-linking network, or multi-responsive integration, the release efficiency decreased significantly under the corresponding conditions, proving that the construction of the responsive layer not only provided the "sensing - feedback" ability but also established an accurate coupling between the component release and the target microenvironment. This "condition-triggered - time-sequential regulation" strategy broke the limitations of passive release of traditional preparations, enabling precise intervention of each functional component at the stage of weed resistance pathway activation.

[0068] In addition, from the measurement results of the encapsulation efficiency and drug loading capacity, it can be seen that the microfluidic core-shell system can more effectively encapsulate easily degradable active substances compared with traditional methods, especially maintaining a high drug loading efficiency when integrating multiple components. This indicates that the multi-component synergistic compounding is not only the integration of functional mechanisms but also forms physical steady-state support at the structural level, avoiding mutual interference or antagonism between components and enhancing the systematic intervention efficiency.

[0069] Test Experiment 2: Experimental samples: The preparation of Example 1, the preparations of Comparative Example 6 and Comparative Example 7.

[0070] Experimental steps: Plant tissue penetration test: Select healthy and growing resistant weeds (such as glyphosate-resistant ryegrass), and take equal-length new leaves (5 cm).

[0071] Treat the leaves of the preparation of Example 1 and Comparative Example 6 with 0.1% Tween-20 as a wetting agent, and spray until completely covered.

[0072] Keep the leaves moist and incubate them in a constant temperature and humidity incubator for 12 hours.

[0073] Take the treated leaves and wash the unpenetrated nanoparticles on the surface with PBS.

[0074] Observe the cross-section through a fluorescence microscope and record the penetration depth of the nanoparticles in the cuticle, epidermal cells, and mesophyll cells.

[0075] Use ImageJ software to quantitatively analyze the fluorescence signal intensity to evaluate the penetration efficiency.

[0076] Lyophilization and rehydration stability test: Take the preparations of Example 1 and Comparative Example 7, perform pre-freezing treatment (-80 °C, 4 hours), and then carry out vacuum lyophilization for 24 hours.

[0077] After obtaining the lyophilized powder, record the powder morphology and color differences respectively.

[0078] Add the lyophilized samples to 10 mL of ultrapure water for rehydration, ultrasonic oscillation for 30 seconds, and then let it stand for 10 minutes.

[0079] Observe the dispersibility of the particles, whether obvious aggregation or precipitation occurs.

[0080] Use DLS to detect the particle size change after rehydration, and measure the PDI value to evaluate the uniformity. The experimental results are shown in Table 2.

[0081] Table 2 Comparison experimental data of plant tissue penetration and lyophilization rehydration stability It can be seen from Table 2 that: On the plant cell wall, the cuticle is usually the most difficult barrier to penetrate. It is composed of a waxy layer and fatty acids, and traditional nanoparticles often have difficulty effectively crossing it. However, the biomimetic penetration peptide significantly improves the penetration depth and intracellular accumulation of nanoparticles by interacting with the plant cell surface. This mechanism effectively breaks through the barrier of the plant cell wall, enabling drugs to enter the plant body more efficiently, thereby enhancing the overall application effect of the preparation.

[0082] During the lyophilization process, trehalose forms a hydrogen bond network to protect the structure of the nanoparticles from being damaged by freezing and drying stresses, avoiding aggregation and precipitation between the particles, and ensuring the particle size and dispersibility after rehydration. In contrast, in Comparative Example 7 without trehalose, obvious aggregation occurred between the lyophilized particles, the particle size increased after rehydration, and the PDI value increased, indicating serious damage to the particle structure during the lyophilization process.

[0083] The biomimetic penetrating peptide improves the targeted penetration ability of drugs and solves the problem of the plant cell wall barrier, while trehalose ensures the stability of the preparation during long-term storage and avoids physical damage.

[0084] Test Experiment 3: Experimental samples: The preparation of Example 1, Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, Comparative Example 5, Comparative Example 6, Comparative Example 8, Comparative Example 9.

[0085] Experimental procedures: Greenhouse experiment design: Select seeds of resistant weeds (such as glyphosate-resistant ryegrass or barnyard grass), sow them evenly in flower pots in the greenhouse, 3 plants per pot, and the soil is a homogeneous culture medium.

[0086] When the weeds grow to the 4-5 leaf stage, start spraying each preparation at a spray volume of 2 mL per pot (containing 5 mg / mL of the active ingredient).

[0087] Use a manual sprayer to keep the leaves evenly covered, and maintain normal light and water supply after spraying.

[0088] Weed control effect determination: Record the yellowing rate and growth inhibition rate of weed leaves 3 days, 7 days, and 14 days after spraying, take pictures with a digital camera, and calculate the yellowing area.

[0089] Harvest the plants 14 days later, measure the fresh weight (g / plant) and record it.

[0090] Calculate the weed control rate (based on the yellowing area) and biomass inhibition rate (based on growth inhibition) of each treatment group. The experimental results are shown in Table 3.

[0091] Table 3 Test data on the control effect of resistant weeds It can be seen from Table 3 that: Glyphosate, as a basic herbicide, is prone to failure due to target mutations or changes in metabolic pathways in traditional use. However, in the present invention, a functionalized drug-loading system containing RG108 and dsRNA is constructed to intervene in the resistance regulation pathway of weeds. RG108 can affect the expression of resistance genes through epigenetic regulation, and dsRNA precisely silences resistance-related genes through the RNA interference mechanism. The two form complementary synergy with glyphosate in terms of time and action path, thus significantly improving the inhibition efficiency of glyphosate in resistant plants.

[0092] Compared with traditional systems that have disordered mixed loading or lack a response layer, the microfluidic construction technology adopted in the present invention endows the formulation with a clear core-shell structure and hierarchical distribution, and integrates dual pH and ROS response modules. In the microenvironment within weeds, this structure can precisely release each component according to different tissues and stress states: glyphosate is rapidly released in an alkaline environment to achieve an initial strike, while RG108 and dsRNA are released delayed in resistant tissues with enhanced oxidative stress to continuously inhibit the activation of resistance pathways.

[0093] In addition, the surface modification of the penetration peptide significantly enhances the ability of the formulation to cross the plant cuticle and cell wall, enabling the active ingredients to accumulate more fully at the target site; while the lyoprotectant maintains the particle size and dispersibility during the long-term storage and transportation of the formulation and the reconstitution process during application, preventing the functional components from inactivating due to physical aggregation. This systematic optimization from structure, transport to release enables the present invention to have excellent adaptability and stability in complex field environments.

[0094] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for controlling glyphosate-potassium salt resistant weeds, characterized in that, It includes the following steps: Prepare a composite composition containing nano-glyphosate potassium salt, resistance reverser and delivery carrier; Embed the composite composition by microfluidic technology to form core-shell structured nanoparticles; Perform surface modification on the nanoparticles to construct environmental response characteristics; Add a biomimetic penetration peptide to the modified nanoparticles and perform freeze-drying stabilization; Control the staged release of each component in the nanoparticles based on environmental parameters; Perform precise spray application according to field conditions.

2. The method for controlling glyphosate-potassium salt resistant weeds according to claim 1, characterized in that, The preparation of the composite composition includes: Ball-mill 280 - 320 parts by weight of glyphosate potassium salt and zirconium beads at a mass ratio of 5:1 - 7:1 for 4 - 6 hours to obtain nano-crystalline glyphosate potassium salt with a D50 of 70 - 90 nm; Mix 45 - 55 parts by weight of propyl 4-chlorophenoxyacetate and 7 - 13 parts by weight of polyacrylic acid to form an oil phase; Add 18 - 22 parts by weight of dsRNA and 8 - 12 parts by weight of RG108 to a ZIF-8 precursor solution composed of 0.2 - 0.3 mol / L Zn(NO3)2 and 0.8 - 1.2 mol / L 2-methylimidazole, and react at room temperature for 2 - 4 hours to form ZIF-8 nanoparticles loaded with dsRNA and RG108; Mix the nano-glyphosate potassium salt, the oil phase and the ZIF-8 nanoparticles to obtain a composite composition containing nano-glyphosate potassium salt, resistance reverser and delivery carrier.

3. The method for controlling glyphosate-potassium salt resistant weeds according to claim 1, characterized in that, The microfluidic technology embedding includes: Use a three-channel chip with a channel width of 180 - 220 μm, and process the composite composition at a flow rate ratio of internal phase:oil phase:external phase = 1:3 - 3.5:4.5 - 5.5; Wherein the internal phase is a glyphosate potassium salt solution containing ethanol / water with a volume ratio of 7:3, and the oil phase contains 80 - 120 parts by weight of PLGA; The external phase contains 0.2 - 0.3 mol / L Zn(NO3)2, and react for 4 - 6 h to form core-shell structured particles.

4. The method for controlling glyphosate-potassium-salt-resistant weeds according to claim 1, wherein, The surface modification includes: Co-incubate 20 - 30 parts by weight of DSPE-PEG2000 with the nanoparticles at 42 - 48 °C for 25 - 35 min; Graft 30 - 40 parts by weight of dimethylaminoethyl methacrylate under ultraviolet light with a wavelength of 360 - 370 nm and an intensity of 8 - 12 mW / cm 2 2 Add 20 - 30 parts by weight of lipoic acid-PEG2000 derivative to construct a ROS-responsive crosslinking network.

5. The method for controlling glyphosate-potassium salt resistant weeds according to claim 1, characterized in that, The freeze-drying stabilization includes: Couple 5 - 7 parts by weight of CPP-EC1 biomimetic penetration peptide to the nanoparticle surface through a thioether bond; Add 30 - 40 parts by weight of trehalose as a freeze-drying protectant; Perform three-stage freeze-drying: pre-freeze at -45 - -40 °C for 4 - 6 h, primary drying at -35 °C / 100 mbar for 8 - 10 h, and secondary drying at 25 °C / 10 mbar for 4 - 6 h.

6. The method for controlling glyphosate-potassium salt resistant weeds according to claim 1, wherein The staged release control includes: Trigger the release of 280 - 320 parts by weight of glyphosate potassium salt when pH > 7.2; Release 30 - 50 parts by weight of S-methylisothiourea sulfate under the condition that the GST enzyme concentration > 0.4 U / mL; Sustainably release 18 - 22 parts by weight of dsRNA@ZIF-8 when the H2O2 concentration > 50 μM.

7. The method for controlling glyphosate-potassium salt resistant weeds according to claim 1, characterized in that, The precise spray application includes: Spray 0.1 mM Ca solution at 200 - 300 L / ha 24 h before applying pesticides; 2+ ​ Control the droplet size VMD = 150 - 200 μm, the spray pressure 3 - 5 bar, and the traveling speed 8 - 10 km / h; Add 0.3 - 0.5% methylated vegetable oil when the environmental humidity < 60%.

8. The method for controlling glyphosate potassium salt-resistant weeds according to claim 3, characterized in that, The microfluidic technology embedding also includes: Emulsify for 8 - 12 min at an oil phase temperature of 35 - 40 °C; Collect the particles with a centrifugal force of 8000 - 10000 × g; Wash 3 times with 200 - 300 parts by weight of ethanol to remove unreacted substances.

9. The method for controlling glyphosate-potassium salt resistant weeds according to claim 4, wherein, The steps of grafting under ultraviolet light include: Control the reaction time for 20 - 30 min to make the polymer swelling ratio reach 2.8 - 3.2; Complete the lipoic acid cross-linking reaction in pH 7.4 PBS buffer for 2 - 4 h.

10. The method for controlling glyphosate-potassium salt resistant weeds according to claim 5, characterized in that, The three-stage freeze-drying includes: Maintain the shelf temperature difference of ±1.5 °C in the pre-freezing stage; The cold trap temperature in the primary drying stage is ≤ -55 °C; The residual moisture after secondary drying is ≤ 1.5%.

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