Preparation method and application of coumoxyl-modified chlorantraniliprole-loaded MOF nano-pesticide

Abstract

This invention discloses a method for preparing and applying a coumaric acid-modified MOF nanopesticide loaded with chlorantraniliprole, belonging to the field of pesticide formulation technology. The nanopesticide comprises: an iron-based metal-organic framework carrier MIL-101(Fe); chlorantraniliprole loaded within the pores of the carrier; and coumaric acid modified on the surface of the carrier through electrostatic interactions. The preparation method includes three steps: MIL-101(Fe) carrier synthesis, chlorantraniliprole loading, and coumaric acid surface modification. The process is simple and the conditions are mild. This invention utilizes the alkaline environment of the fall armyworm's gut to trigger carrier degradation, achieving targeted and controlled release of active molecules. Simultaneously, the released MIL-101(Fe) is reduced under the action of GSH in the gut, generating reactive oxygen species through the Fenton reaction, forming a synergistic insecticidal effect with coumaric acid. Experiments have shown that this nanopesticide exhibits far superior insecticidal activity against fall armyworm compared to single agents or carriers, possessing excellent insecticidal activity and environmental compatibility, making it suitable for green agricultural production.

Description

Technical Field

[0001] This invention relates to the field of pesticide formulation technology, specifically to a method for preparing and applying a coumaric acid-modified MOF nanopesticide loaded with chlorantraniliprole, which is particularly suitable for the green control of fall armyworm. Background Technology

[0002] The fall armyworm (Spodoptera frugiperda), a global invasive pest, is characterized by its high reproductive capacity, rapid migration, and wide host range, causing serious damage to crops such as corn. Currently, the control of the fall armyworm mainly relies on chemical pesticides. However, the long-term and excessive use of chemical pesticides can easily lead to problems such as pesticide resistance in pests, environmental pollution, and pesticide residues in agricultural products, which does not meet the needs of green agriculture development.

[0003] Corn contains natural insecticidal active molecules such as coumaric acid and ferulic acid, which have advantages such as good environmental compatibility and low resistance to pesticide development in pests. However, these active molecules also have drawbacks such as poor water solubility, easy degradation, and short duration of action, which limit their direct application. Existing nanopesticide carriers mostly use inorganic nanoparticles or polymer materials, which have problems such as low pesticide loading, lack of targeted controlled release capability, and single insecticidal mechanism. They are also difficult to adapt to the special microenvironment of the fall armyworm's gut (pH 8.5-10), which is alkaline, and cannot achieve precise and efficient release of active molecules.

[0004] Iron-based metal-organic framework (MOF) materials such as MIL-101(Fe) possess high specific surface area, tunable pore size, and good biocompatibility. Their structural stability is significantly affected by pH, readily undergoing coordination bond dissociation in alkaline environments, offering potential for targeted controlled release in the gut. However, how to organically combine natural insecticidal active molecules with chemical pesticides and utilize the properties of MOF materials to construct responsive release systems targeting the gut environment of specific pests, while simultaneously leveraging the synergistic insecticidal effect of multiple components, remains a pressing technical challenge in this field. Currently, there are no reports on using coumaric acid-modified MIL-101(Fe) as a carrier to load chlorantraniliprole and designing nanopesticides specifically for the gut environment of fall armyworm. Therefore, there is an urgent need to develop novel nanopesticide formulations that combine targeted controlled release and synergistic insecticidal functions. Summary of the Invention

[0005] To address the technical problems of existing chemical pesticides polluting the environment, strong pesticide resistance in pests, limited application of natural insect-resistant active molecules, and lack of targeted controlled release capabilities of existing nanocarriers, this invention provides a method for preparing and applying a coumaric acid-modified MOF nanopesticide loaded with chlorantraniliprole, achieving efficient and green control of fall armyworm.

[0006] In a first aspect, the present invention provides an iron-based MOF nanopesticide, comprising:

[0007] Iron-based metal-organic framework carrier MIL-101(Fe);

[0008] Chlorantraniliprole loaded within the pores of the carrier;

[0009] p-Coumaric acid modified on the surface of the carrier through electrostatic interaction;

[0010] The electrostatic interaction is characterized by the change of the Zeta potential from negative to positive before and after modification.

[0011] Secondly, the present invention provides a method for preparing the above-mentioned iron-based MOF nanopesticide, comprising the following steps:

[0012] (1) Synthesis of MIL-101(Fe) support;

[0013] (2) Chlorantraniliprole was loaded into the MIL-101(Fe) support to obtain CAP@MIL-101(Fe);

[0014] (3) Mix p-coumaric acid aqueous solution with CAP@MIL-101(Fe) and stir in the dark to modify the surface, so as to obtain CAP@MIL-101(Fe)-PCA.

[0015] Thirdly, the present invention provides the application of the above-mentioned iron-based MOF nanopesticide in the control of lepidopteran pests.

[0016] Fourthly, the present invention provides a method for controlling fall armyworm by applying the above-mentioned MOF nanopesticide to crops or the environment.

[0017] Beneficial effects

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] (1) Excellent targeted controlled release performance: The degradation of the MIL-101(Fe) carrier is triggered by the alkaline environment (pH 8.5-10) in the intestine of the fall armyworm, achieving precise release of chlorantraniliprole and p-coumaric acid. Experiments show that the release rate is close to 70% after 24 hours at pH 10.0, while the release rate is only close to 20% after 24 hours at pH 6 and pH 7, significantly reducing drug loss in non-target environments and improving utilization efficiency.

[0020] (2) Multiple synergistic insecticidal mechanisms: This invention constructs a triple synergistic system of "chemical pesticide - natural active molecule - carrier functionalization". The combination of coumaric acid and chlorantraniliprole significantly enhances insecticidal activity; simultaneously, the MIL-101(Fe) carrier releases... Under the action of high concentrations of glutathione (GSH) in the insect's intestines, it is reduced to The Fenton reaction generates a large amount of reactive oxygen species (ROS), which damage the structure of intestinal epithelial cells. Experiments have shown that MIL-101(Fe) can consume GSH and generate ROS in a concentration- and time-dependent manner, forming a synergistic insecticidal effect with the active molecules. The insecticidal activity of the nano-pesticide treatment group against fall armyworm is far superior to that of single agents or carrier groups.

[0021] (3) Good environmental compatibility: The carrier MIL-101(Fe) has excellent biocompatibility. Iron is one of the essential metal elements for plant growth. The surface-modified p-coumaric acid is a natural product of corn and is safe for beneficial insects and crops. The release of the material in non-target environments is limited, reducing environmental risks.

[0022] (4) Simple preparation process: The three-step synthesis process has mild conditions (room temperature to 110℃), requires no special equipment, has low production costs, and is suitable for large-scale application. By adjusting... The ratio of salt to organic ligands and the loading temperature can be used to precisely control the loading rate of chlorantraniliprole, which can reach more than 85%. Attached Figure Description

[0023] Figure 1 These are the experimental results of the cytotoxicity of maize secondary metabolites to coumaric acid according to the present invention;

[0024] Figure 2 This invention is for the determination of the inhibitory activity of coumaric acid on Sf9 cells;

[0025] Figure 3 This invention aims to determine the bioactivity of coumaric acid and chlorantraniliprole in newly hatched larvae after 48 hours and the mortality rate of larvae under different treatments over time.

[0026] Figure 4 The following are characterization images of the MIL-101(Fe) material of this invention: (a), (b) scanning electron microscope image of MIL-101(Fe), and (c) X-ray diffraction pattern of MIL-101(Fe);

[0027] Figure 5 The average particle size of MIL-101(Fe) in different solvents is shown.

[0028] Figure 6 The images show the FT-IR spectra of MIL-101(Fe), CAP@MIL-101(Fe), and CAP@MIL-101(Fe)-PCA before and after modification, as well as (b) the hydrated particle size distribution and (c) the potential diagram.

[0029] Figure 7XPS scan spectra of MIL-101(Fe), CAP@MIL-101(Fe) and CAP@MIL-101(Fe)-PCA of the present invention;

[0030] Figure 8 This invention evaluates the time- and concentration-dependent GSH consumption of MIL-101(Fe).

[0031] Figure 9 This invention evaluates the time- and concentration-dependent ROS generation by MIL-101(Fe).

[0032] Figure 10 The release curves of CAP@MIL-101(Fe)-PCA of the present invention under different pH conditions;

[0033] Figure 11 The bioactivity of iron-based materials with different modifications according to the present invention against Spodoptera litura and Spodoptera litura was determined.

[0034] Figure 12 The present invention describes the effects of different modified iron-based materials on Sf9 cytotoxicity and IC50. 50 . Detailed Implementation

[0035] To enable those skilled in the art to better understand the technical solutions of this invention, the present application will be further described in detail below with reference to embodiments.

[0036] The three-step process of MIL-101(Fe) carrier synthesis, p-coumaric acid loading, and surface modification is simple, mild, and suitable for large-scale production; by controlling... Parameters such as the salt-to-organic ligand ratio and drug loading temperature enable precise control of the active molecule loading rate. During application, after the fall armyworm ingests the nano-pesticide, the coordination bonds of the MIL-101(Fe) carrier dissociate in the alkaline environment of the intestine (pH 8.5-10), releasing p-coumaric acid. Simultaneously, the Fe³⁺ released by the carrier is reduced to [a specific compound] under the action of GSH in the intestine. It generates a large amount of reactive oxygen species through the Fenton reaction, which damages the structure of intestinal epithelial cells and forms a synergistic insect-resistant effect with active molecules.

[0037] Example 1: Screening and Activity Prediction of Secondary Metabolites in Maize

[0038] Ninety-two maize secondary metabolites were collected from maize roots, stems, leaves, and bran. Four inexpensive and readily available compounds were selected: 4-methoxycinnamic acid, methyl trans-ferulate, trans-caryophyllene, and p-coumaric acid. The insecticidal activity of these four maize secondary metabolites was predicted using a chemical agricultural (insecticide) activity prediction website developed by China Agricultural University. A higher APPi-score (applied to 1) indicated better insecticidal activity. The results are shown in Table 1. 4-methoxycinnamic acid exhibited the best insecticidal activity, while p-coumaric acid, with an APPi-score of 0.8269, demonstrated good insecticidal potential.

[0039] Table 1. Prediction of insecticidal activity of maize secondary metabolites

[0040]

[0041] Example 2: Cytotoxicity test of coumaric acid

[0042] 1. Cell Culture

[0043] Sf9 cells (derived from the ovarian tissue of the fall armyworm) were cultured in Sf-900 medium containing 10% serum and 1% penicillin antibiotics at a standard culture environment of 28°C. When the cell density reached more than 80%, the cells were pipetted and passaged.

[0044] 2. Cytotoxicity assay

[0045] Cells were counted after resuspending, at a density of 8 × 10⁸ cells per well. 4 Cells were seeded at a density of 100 µL in each well of a 96-well plate. The cells were incubated overnight. The culture medium was then aspirated, and medium containing different concentrations (37.5 μM, 75 μM, 150 μM, 300 μM, 600 μM, 1200 μM) of p-coumaric acid was added. The cells were incubated for another 24 h. Three replicates were performed for each concentration.

[0046] After incubation, the culture medium was aspirated, and medium containing Cell Counting Kit-8 (CCK-8) solution was added (CCK-8: medium = 1:10), and incubation continued for 2 h. The absorbance at 450 nm was measured using a microplate reader to assess the cytotoxicity to coumaric acid.

[0047] 3. Experimental Results

[0048] Cytotoxicity data such as Figure 1 As shown, after 24 h of co-culture, the survival rate of Sf9 cells treated with coumaric acid decreased in a concentration-dependent manner, with the cell survival rate in the high-concentration (1200 μM) treatment group decreasing to 18.9%. The half-maximal inhibitory concentration (IC50) of coumaric acid on Sf9 cells was calculated by logarithmic fitting.50 The value was 215.28 μM. Figure 2 The results showed that p-coumaric acid has a certain degree of toxicity to fall armyworm cells.

[0049] Example 3: Bioactivity experiment of p-coumaric acid against fall armyworm

[0050] 1. Insect rearing

[0051] Fall armyworm test insects were reared in the laboratory at 27±1℃, photoperiod L14:D10, and relative humidity 70%. Adult insects were placed in square rearing cages with a diameter of 35 cm for mating and fed 10% honey water. Egg masses were collected daily, and the hatched larvae were fed artificial feed.

[0052] 2. Bioactivity assay

[0053] The bioactivity assay is based on Chapter 3, "Indoor Bioassay Methods for Insecticides - Mixed-Poison Feed Method," in the book "Pesticide Bioassay" published by China Agriculture Press.

[0054] (1) After mixing artificial feed with hot water, dispense 900 μL into each well of a 24-well plate using a pipette;

[0055] (2) Prepare p-coumaric acid to concentrations of 200 mg / L and 500 mg / L with pure water, and prepare chlorantraniliprole to concentration of 1 mg / L with pure water. Chlorantraniliprole is compounded with p-coumaric acid of different concentrations to form compound insecticidal solutions, each solution being 30 mL.

[0056] (3) Use a pipette to drop compounds of different concentrations onto the surface of the feed in a 24-well plate, 100 μL per well;

[0057] (4) Place the 24-well plate in a dry environment until the compound completely impregnates the feed and the surface remains dry;

[0058] (5) Third-instar larvae of fall armyworm were placed in feed well plates containing the compound and fed with 3 biological replicates for each compound concentration, with 30 larvae per replicate.

[0059] (6) Record the number of surviving insects and their age for each group of test insects every day, and calculate the mortality rate.

[0060] Meanwhile, a treatment group was set up with a combination of coumaric acid and chlorantraniliprole to explore the synergistic effect of the two.

[0061] 3. Experimental Results

[0062] Bioactivity assay results as follows Figure 3As shown, the mortality rate of the control group was approximately 9.1% on day 7, indicating a low natural mortality rate. Over time, the mortality rate of the coumaric acid-treated group was consistently higher than that of the control group. Figure 3 (a, g) The mortality rate in the high-concentration treatment group was higher than that in the low-concentration treatment group, reflecting both concentration-dependent and time-dependent effects. Figure 3 bc, g). On day 7, the mortality rate in the high-concentration (500 mg / L) coumaric acid treatment group was approximately 38.2%, indicating that high concentrations of coumaric acid possess certain gastric toxicity activity.

[0063] It is noteworthy that on day 3, the insecticidal activity of the 500 mg / L p-coumaric acid (PCA) combined with chlorantraniliprole (CAP) treatment group was significantly higher than that of the chlorantraniliprole alone group. Figure 3 (df), indicating that the two have a good synergistic effect.

[0064] Example 4: Preparation and Characterization of MIL-101(Fe) Material

[0065] 1. Synthesis of MIL-101(Fe) support

[0066] Weigh out 3.24 g 0.664 g of 2-aminoterephthalic acid was dispersed in 60 mL of N,N-dimethylformamide and ultrasonically dispersed for 10 min. The mixture was then transferred to a reaction vessel and stirred at 500 rpm for 30 min at room temperature. The reaction was then carried out at 110 °C for 20 h. After the reaction, the mixture was cooled to room temperature. The product was transferred to a 50 mL centrifuge tube and centrifuged at 8000 rpm for 5 min. The product was washed three times with DMF and once with anhydrous ethanol. The precipitate was collected by centrifugation and washed three times sequentially with N,N-dimethylformamide and ethanol. The product was then vacuum dried at 80 °C for 8 h to obtain the MIL-101(Fe) supported product.

[0067] 2. Preparation of iron-based nanopesticides loaded with chlorantraniliprole

[0068] 0.5 g of chlorantraniliprole was dissolved in 50 mL of N,N-dimethylformamide, and 0.5 g of the MIL-101(Fe) support prepared in Example 4.1 was added. The mixture was stirred at 500 rpm for 0.5 h at 25 °C. The mixture was then transferred to a reaction vessel and reacted at 110 °C for 20 h. After the reaction was complete, the mixture was cooled to room temperature, centrifuged to collect the precipitate, washed three times successively with N,N-dimethylformamide and ethanol, and dried under vacuum at 80 °C for 8 h to obtain CAP@MIL-101(Fe) iron-based nanopesticide. The chlorantraniliprole loading rate was determined to be 85.8%.

[0069] 3. Preparation of coumaric acid-modified iron-based nanopesticides

[0070] Weigh 0.1 g of CAP@MIL-101(Fe) and p-coumaric acid and dissolve them separately in 10 mL of water. Add CAP@MIL-101(Fe) dropwise to the p-coumaric acid solution while stirring continuously in the dark at 300 rpm for 24 h. After centrifugation, wash the product twice with pure water and once with anhydrous ethanol, then dry at 80℃ to obtain CAP@MIL-101(Fe)-PCA.

[0071] 5. Characterization of MIL-101(Fe) material

[0072] (1) Crystal structure characterization

[0073] The synthesized MIL-101(Fe) was characterized using X-ray diffraction. The results showed that ( Figure 4 c) All characteristic diffraction peaks are in high agreement with the standard card of MIL-101(Fe), indicating that the synthesized MIL-101(Fe) has the expected crystal structure.

[0074] (2) Morphological characteristics

[0075] Transmission electron microscopy (TEM) images show ( Figure 4 (ab), the synthesized MIL-101(Fe) has a regular octahedral structure.

[0076] (3) Measurement of hydrated particle size

[0077] 1) Sample preparation: Take an appropriate amount of material and disperse it in ultrapure water, and sonicate it for 10 min to obtain a uniform dispersion.

[0078] 2) Instrument calibration: The Zetasizer Nano ZS90 was calibrated using standard particle size samples.

[0079] 3) Particle size determination: Add the dispersion to a cuvette, set the measurement parameters, and measure the hydrated particle size at room temperature. Repeat 3 times and take the average value.

[0080] The results show that ( Figure 5 MIL-101(Fe) exhibits uniform hydration particle size distribution in PBS, Sf-900 medium, and water, conforming to a normal distribution, and demonstrates good solubility and stability. Figure 6 This indicates that the material has good morphological uniformity.

[0081] (4) Determination of Zeta potential of AP@MIL-101(Fe)-PCA surface modification

[0082] 1) Sample preparation: Take an appropriate amount of material and disperse it in ultrapure water, and sonicate it for 10 min to obtain a uniform dispersion.

[0083] 2) Instrument calibration: The Zetasizer Nano ZS90 was calibrated using standard particle size samples.

[0084] 3) Potential measurement: Replace the sample cell, inject the same batch of dispersion, and measure the zeta potential before and after modification. Repeat 3 times and take the average value.

[0085] MIL-101(Fe) was surface-modified using PCA via electrostatic interaction. Infrared spectroscopy analysis showed that the modified CAP@MIL-101(Fe)-PCA retained the characteristic absorption peaks of both PCA and MIL-101(Fe). Figure 6 a) indicates that PCA successfully bonded to MIL-101(Fe). Zeta potential testing results show that the surface potential of the modified material changed from approximately -5 mV to approximately +19 mV (a). Figure 6 c), this significant potential change confirms that p-coumaric acid was successfully modified onto the support surface via electrostatic interactions. Furthermore, the modified CAP@MIL-101(Fe)-PCA exhibited good dispersibility and stability, and its hydrated particle size did not change significantly before and after modification. Figure 6 b). The above results collectively demonstrate that the CAP@MIL101-(Fe)-PCA material loaded with CAP and modified with PCA was successfully synthesized.

[0086] (5) XPS characterization of MIL-101(Fe), CAP@MIL-101(Fe) and CAP@MIL-101(Fe)-PCA

[0087] Weigh 20 mg of MIL-101(Fe), CAP@MIL-101(Fe) and CAP@MIL-101(Fe)-PCA and place them in a 1.5 mL centrifuge tube for XPS testing.

[0088] XPS full spectrum ( Figure 7 a) The presence of elements such as Fe, C, N, and O was confirmed. The differences in peak intensity among different materials reflected variations in elemental content, indirectly proving the successful modification of MIL-101 (Fe) by chlorantraniliprole and p-coumaric acid. XPS spectra of Fe 2p ( Figure 7 bd) shows that MIL-101(Fe), CAP@MIL-101(Fe), and CAP@MIL-101(Fe)-PCA contain and After peak fitting, the characteristic peaks indicate that iron is mainly composed of... The presence of valence states, along with slight shifts in the Fe 2p binding energy and peak shape across different materials, reflects the interaction between Fe and CAP and PCA, demonstrating that the modification process altered the chemical structure of the materials. XPS spectra of Cl 2p (… Figure 7 e) Characteristic peaks appear only in CAP@MIL-101(Fe)-PCA, and The signal corresponds to the components of CAP, directly confirming that CAP has been successfully loaded into the composite material.

[0089] The above results collectively demonstrate the successful synthesis of CAP@MIL-101(Fe)-PCA nanopesticides loaded with chlorantraniliprole and surface-modified with p-coumaric acid. Furthermore, the influence of the modification process on the chemical environment of the material was clarified, providing a structural basis for subsequent analysis of the material's properties.

[0090] Example 5: Performance Testing of MIL-101(Fe) Material

[0091] 1. Glutathione (GSH) consumption capacity determination

[0092] Excess glutathione (GSH), a core endogenous antioxidant and detoxifying component in the gut of the fall armyworm, can enhance the larvae's tolerance to plant secondary metabolites and exogenous stress by scavenging exogenous reactive oxygen species (ROS) and regulating glutathione S-transferase (GST) activity. It serves as a key antioxidant barrier for the fall armyworm's adaptation to host defense. Therefore, targeting and depleting GSH in the fall armyworm's gut to disrupt its antioxidant defense system is a core strategy for enhancing the insecticidal efficacy of MIL-101(Fe).

[0093] MIL-101(Fe) material is rich in Coordination unsaturation sites and / The ability to switch valence states provides key theoretical support for its efficient consumption of GSH in the gut microenvironment of the fall armyworm: the gut of the fall armyworm is a slightly alkaline environment, which can promote the conversion of GSH in the MIL-101(Fe) skeleton. Partial dissociation occurs, and the thiol group (-SH) in the GSH molecule has strong reducing properties and can react with the dissociated group. A specific redox reaction occurs—GSH is oxidized to oxidized glutathione (GSSG). It is then restored to Meanwhile, the generated It can further form a stable -SH group with GSH. -GSH coordination complexes achieve continuous consumption of GSH in the gut through a dual mechanism of redox reaction and coordination chelation. This process not only breaks down the antioxidant barrier in the larval gut, but the accumulated Fe²⁺ can also generate a large number of hydroxyl radicals in the gut via the Fenton reaction. This damages the structure and function of intestinal epithelial cells, ultimately inhibiting the growth and development of larvae.

[0094] The specific steps are as follows:

[0095] (1) DTNB was selected as a probe to detect the material’s ability to consume GSH.

[0096] (2) To detect its concentration dependence, different concentrations of the material (0, 10, 15, 20, 25, 30 μg / mL) and GSH (1×10⁻⁶) were prepared. -3 Mix the 100 μL (mol / L) solution in PBS at room temperature and incubate for 6 h. Then dilute 100 μL of the mixture to 1 mL with PBS and add 2 × 10⁻⁶ DTNB. -3 The -SH of the remaining GSH was detected by measuring the change in its absorption spectrum at 412 nm (mol / L).

[0097] (3) To detect its time dependence, two equal concentrations of the material (25 μg / mL) and GSH (1×10⁻⁶) were prepared respectively. -3 Mix the solution (mol / L) in PBS at room temperature. At different time points (0, 0.5, 1, 2, 4, 6 h), take 100 μL of the mixture and dilute it to 1 mL with PBS. Then add DTNB (2×10⁻⁶ mol / L). -3 The -SH of the remaining GSH was detected by measuring the change in its absorption spectrum at 412 nm (mol / L).

[0098] The results are as follows Figure 8 The results showed that the characteristic absorption peak of DTNB at 412 nm decreased significantly in a concentration-dependent manner with increasing MIL-101(Fe) concentration. To further verify the GSH consumption effect of MIL-101(Fe), this invention also tested the sustained GSH consumption capacity of MIL-101(Fe) at different incubation times. The results showed that with prolonged incubation time, the absorption peak of DTNB at 412 nm in MIL-101(Fe) treated GSH decreased significantly ( Figure 8 The above results indicate that MIL-101(Fe) can be passed through... / The mediated thiol-targeting response achieves efficient and selective GSH consumption in the gut microenvironment of the fall armyworm, not only breaking down the larval antioxidant defense barrier but also simultaneously activating... The mediated oxidative damage effect lays a key foundation for enhancing its control efficacy against fall armyworm.

[0099] 2. Determination of Reactive Oxygen Species (ROS) Generation Capacity

[0100] The fall armyworm's gut microenvironment exhibits a typical "redox balance defense system"—endogenous hydrogen peroxide (H2O2) is produced during intestinal epithelial cell metabolism, while a high concentration of glutathione (GSH) in the gut acts as a core antioxidant barrier, effectively scavenging reactive oxygen species (ROS) and reducing exogenous oxidative damage. This helps larvae tolerate host plant secondary metabolites and environmental stresses, which is a key mechanism for maintaining intestinal homeostasis and resisting exogenous insecticides. Therefore, disrupting the redox balance in the fall armyworm's gut and amplifying ROS accumulation is a core strategy for enhancing the insecticidal efficacy of MIL-101 (Fe).

[0101] The specific steps are as follows:

[0102] (1) MB was selected as a probe to detect the generation of ROS under the action of the material.

[0103] (2) Prepare materials of different concentrations (0, 10, 15, 20, 25, 30 μg / mL) and H2O2 (1×10⁻⁶) respectively. -3 mol / L) and TMB (5×10 -4 The mixture (mol / L) was mixed in PBS solution at room temperature and incubated at room temperature for 6 h. Then, 100 μL of the mixture was diluted to 1 mL with PBS solution, and the absorption spectrum change at 660 nm was detected to detect the ROS generation capacity.

[0104] (3) To further investigate the material's ability to generate ROS under conditions of excessively high GSH levels, two equal concentrations of the material (25 μg / mL) and H2O2 (1×10⁻⁶) were prepared. -3 mol / L), TMB (5×10 -4 mol / L) and GSH (1×10 -3 The mixture (mol / L) was mixed in PBS solution at room temperature and incubated at room temperature for 6 h. Then, 100 μL of the mixture was diluted to 1 mL with PBS solution, and the absorption spectrum change at 660 nm was detected to detect the ROS generation capacity.

[0105] The results are as follows Figure 9 As shown, with increasing MIL-101(Fe) concentration gradient, the characteristic absorption peak of TMB at 660 nm decreased significantly in a concentration-dependent manner, and the decrease was greater at higher concentrations, indicating that MIL-101(Fe) can efficiently and dose-dependently generate ROS. Further extending the incubation time (the retention process of the material in the intestine after larval ingestion) confirmed the time-dependent nature of its ROS generation. Even in the presence of GSH, MIL-101(Fe) could still effectively generate ROS, indicating that the material can overcome the pest's antioxidant defense system and exert an oxidative damage effect.

[0106] Example 6: pH-responsive release performance test of CAP@MIL-101(Fe)-PCA

[0107] 1. Preparation of release medium

[0108] Phosphate buffer solutions with pH 10.0 (simulating the alkaline environment of the fall armyworm gut), pH 7.0 (simulating the natural environment), and pH 6.0 (simulating the slightly acidic environment) were prepared as release media for later use.

[0109] 2. Dialysis bag pretreatment

[0110] Select dialysis bags with matching molecular weight cutoffs, activate them in advance, and then rinse them with release medium of the corresponding pH for later use.

[0111] 3. Release experiment

[0112] Accurately weigh 20 mg of CAP@MIL-101(Fe)-PCA material and place it into a pretreated 3500 Da dialysis bag. Add a small amount of the corresponding pH release medium to moisten the bag, then seal it to prevent leakage. Immerse the sealed dialysis bag in a container containing 200 mL of the corresponding pH release medium. Place the container in a constant temperature shaker, set the temperature to 28℃ (simulating the intestinal temperature of the fall armyworm), and the rotation speed to 150 rpm. Start the shaking.

[0113] At time points of 0, 5, 10, 15, 20, and 25 h, 5 mL of supernatant was accurately pipetted from each system, and 5 mL of fresh release medium corresponding to the corresponding pH was immediately added. The concentration of p-coumaric acid in the samples at each time point was determined by high-performance liquid chromatography (HPLC), and the cumulative release rate was calculated. Three parallel experiments were set up for each pH system, and the average release rate was taken. Finally, CAP release curves were plotted under different pH conditions.

[0114] 4. Experimental Results

[0115] Release curve as shown Figure 10 The figure shows the release behavior under three environments: pH 10, pH 7, and pH 6. In the initial stage (approximately 0-10 h), the CAP release rate under all three pH conditions increased with time. In the subsequent stage, the release rate under pH 6 and pH 7 conditions gradually leveled off and remained at approximately 30%, while the release rate under pH 10 conditions continued to rise, reaching nearly 70% at 25 h. This result indicates that the material release is pH-responsive, with more complete release in alkaline environments and limited release in neutral and weakly acidic environments. This characteristic is compatible with the alkaline environment of the midgut of lepidopteran insects. This suggests that the material can efficiently release the active ingredient in the midgut environment of pests, which is beneficial for improving insecticidal efficacy.

[0116] Example 7: Determination of the bioactivity of iron-based MOF nanopesticides against fall armyworm and beet armyworm.

[0117] 1. Insect rearing

[0118] Fall armyworm and beet armyworm were reared in the laboratory at 27±1℃, photoperiod L14:D10, and relative humidity 70%. Adults were placed in square rearing cages with a diameter of 35 cm for mating and fed 10% honey water. Egg masses were collected daily, and the hatched larvae were fed artificial feed.

[0119] 2. Bioactivity assay

[0120] The experimental methods for bioactivity determination are based on Chapter 3 of "Pesticide Bioassay" published by China Agriculture Press - Indoor Bioassay Methods for Insecticides - Mixed Poison Feed Method.

[0121] (1) Mix the artificial feed with hot water and dispense it into 24-well plates, 900 μL per well;

[0122] (2) Prepare MIL-101(Fe), CAP@MIL-101(Fe) and CAP@MIL-101(Fe)-PCA to a concentration of 5 μM using an aqueous solution containing 0.1% Tween-80. Prepare CAP and PCA to a concentration of 5 μM using pure water as controls. 30 mL of each solution.

[0123] (3) Use a pipette to drop compounds of different concentrations onto the surface of the feed in a 24-well plate, 100 μL per well;

[0124] (4) Place the 24-well plate in a dry environment until the compound drug is completely impregnated in the feed and the surface is kept dry;

[0125] (5) First-instar larvae were fed in feed well plates containing the compound, with three biological replicates for each compound concentration and 30 larvae per replicate.

[0126] (6) Record the number of surviving insects in each group every day, observe for 7 consecutive days, and calculate the survival rate.

[0127] 3. Experimental Results

[0128] Bioactivity assay results as follows Figure 11As shown, the 7-day survival rate curves under different treatment groups correspond to different treatments (DMSO as control, CAP as chlorantraniliprole, MIL-101(Fe) as carrier, PCA, etc. as p-coumaric acid, and various composite material systems): The survival rate of the control group DMSO remained close to 100%. The survival rate of the single-component (CAP, MIL-101(Fe), PCA) treatment groups decreased relatively slowly, while the survival rate of the CAP@MIL-101(Fe) and CAP@MIL-101(Fe)-PCA composite nanomaterial treatment groups decreased significantly faster. Among them, the CAP@MIL-101(Fe)-PCA material showed good insecticidal effects against both Spodoptera litura and Spodoptera litura. The above results indicate that the modified MIL-101(Fe)-based composite nanomaterials have far superior insecticidal activity against these two pests compared to single agents or carriers, and composite materials can significantly improve the effect.

[0129] Example 8: Toxicity experiment of iron-based MOF nanopesticides on Sf9 cells

[0130] 1. Cell Culture

[0131] Insect cells from the ovarian tissue of female Sf9 fall armyworm were cultured in Sf-900 medium containing 10% serum and 1% antibiotics at a standard culture environment of 28°C. When the cell density reached more than 80%, the cells were pipetted and passaged.

[0132] 2. Cytotoxicity assay

[0133] Cells were counted after resuspending, at a density of 1 × 10⁶ cells per well. 4 Cells were seeded at a density of 100 µL in each well of a 96-well plate. The cells were incubated overnight. The culture medium was then aspirated, and different concentrations (9.375, 18.75, 37.5, 75, 125, 500 µg / L) of MIL-101(Fe), CAP@MIL-101(Fe), CAP@MIL-101(Fe)-PCA, CAP, and PCA solutions were added, and the cells were incubated for another 24 h.

[0134] After incubation, the culture medium was aspirated, and culture medium containing Cell Counting Kit-8 (CCK-8): medium = 1:10 was added, and incubation continued for 2 h. The absorbance at 450 nm was measured using a microplate reader to assess the cytotoxicity of different treatments.

[0135] 3. Experimental Results

[0136] The cytotoxicity data are as follows ( Figure 12After 24 hours of co-culture, the relative cell viability of the single-component treatment groups (CAP, PCA, MIL-101(Fe)) decreased gradually at each concentration, and remained at a high level even at high concentrations. However, the cell viability of the CAP@MIL-101(Fe) and CAP@MIL-101(Fe)-PCA composite nanomaterials decreased significantly with increasing concentration, approaching zero at high concentrations. 50 (Half-inhibitory concentration) is lower. These results indicate that the modified CAP@MIL-101(Fe)-PCA composite nanomaterials exhibit high cytotoxicity against Sf9 cells, with an IC50 value of [missing value]. 50 It is 0.123 mg / L ( Figure 12 The concentrations of the composite nanomaterials were significantly lower than those of the single component. The results indicate that the composite nanomaterials exhibit high toxicity to Sf9 cells, corresponding to their insecticidal effect on live pests, thus verifying that the material exerts its insecticidal effect by inhibiting pest cell activity.

Claims

1. An iron-based MOF nanopesticide, characterized in that, include: Iron-based metal-organic framework carrier MIL-101(Fe); Chlorantraniliprole loaded within the pores of the carrier; p-Coumaric acid modified on the surface of the carrier through electrostatic interaction; The electrostatic interaction is characterized by the change of the Zeta potential from negative to positive before and after modification.

2. The iron-based MOF nanopesticide according to claim 1, characterized in that, The MIL-101(Fe) carrier is composed of It is prepared by a solvothermal method with 2-aminoterephthalic acid.

3. The iron-based MOF nanopesticide according to claim 2, characterized in that, The loading rate of chlorantraniliprole is above 85%.

4. A method for preparing iron-based MOF nanopesticides as described in any one of claims 1-3, characterized in that, Includes the following steps: (1) Synthesis of MIL-101(Fe) support; (2) Chlorantraniliprole was loaded into the MIL-101(Fe) support to obtain CAP@MIL-101(Fe); (3) Mix p-coumaric acid aqueous solution with CAP@MIL-101(Fe) and stir in the dark to modify the surface, so as to obtain CAP@MIL-101(Fe)-PCA.

5. The method according to claim 4, characterized in that, Step (2) involves loading the drug into N,N-dimethylformamide and reacting it at 110°C for 20 h.

6. The method according to claim 4, characterized in that, In step (3), the stirring time is 24 h and the stirring speed is 300 rpm.

7. The application of an iron-based MOF nanopesticide as described in any one of claims 1-3 or an iron-based MOF nanopesticide prepared by the method described in any one of claims 4-6 in the control of fall armyworm.

8. The application according to claim 7, characterized in that, The iron-based MOF nanopesticide achieves targeted release of chlorantraniliprole and p-coumaric acid in the alkaline environment of the fall armyworm's gut; simultaneously, the MIL-101(Fe) carrier releases... Reduced to its original form in the insect's gut. It generates reactive oxygen species through the Fenton reaction, which, together with p-coumaric acid, act on the fall armyworm.

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