Application of chlorantraniliprole and imidacloprid loaded double-controlled-release nano-carrier in improvement of prevention and control effect of lissorhoptrus oryzophilus

Abstract

The invention discloses application of a chlorantraniliprole and imidacloprid loaded double-controlled-release nano-carrier to improvement of the prevention and control effect of lissorhoptrus oryzophilus. The nano pesticides CAP (at) MSN-SS-CMCS and IMI (at) MSN-SS-CMCS disclosed by the invention can be used for continuously and effectively preventing and controlling the lissorhoptrus oryzophilus and reducing the harm of the lissorhoptrus oryzophilus. By using the modified nano pesticide, the use amount of the raw pesticide can be effectively reduced, and the pollution and poison of the pesticide to the environment are reduced. The nano pesticides CAP-coated MSN-SS-CMCS and IMI-coated MSN-SS-CMCS are harmless to the growth of rice, have no phytotoxicity phenomena such as leaf rolling, leaf burning and yellowing, and have good safety to zebra fish, and the toxicity of the nano pesticides is far smaller than that of original drugs.

Description

Technical Field

[0001] The present invention belongs to the field of nanopesticides, and particularly relates to the application of a dual-controlled-release nanocarrier loaded with chlorantraniliprole and imidacloprid in improving the control effect of rice water weevils. Background Art

[0002] Pesticides play a vital role in traditional agriculture, not only significantly increasing crop yields to meet growing food demands but also playing a key role in sustainable agricultural development. However, in practice, over 90% of pesticides fail to reach their target crops, instead being lost through volatilization, drift, photodegradation, slippage, and rainwater washoff. Excessive or inappropriate use of pesticides can pose a serious threat to crop safety. These chemicals are not only toxic to non-target organisms but can also accelerate the evolution of resistance in target organisms, ultimately leading to ecosystem damage and environmental pollution, posing a serious threat to human health.

[0003] The rice water weevil (Lissorhoptrus oryzophitus) is the only quarantined coleopteran pest in my country's rice production. It seriously affects rice yields and may even cause large-scale yield reductions or even complete crop failures. The adult rice water weevil feeds on the upper epidermis and mesophyll of young rice leaves, hindering the photosynthesis of rice leaves, while the larvae feed on the roots of rice, causing the roots of the plants to break and fall over. Currently, the most effective control measure for rice water weevils is still chemical control, and the best control time is the adult stage. However, adults only stay on rice leaves for 2 to 3 weeks, after which they gradually dive into the roots of rice to lay eggs. In addition, with the exception of Guangdong Province, where two generations occur a year, rice water weevils in other provinces only have one generation a year. The short control window, difficult monitoring, and strong drug resistance significantly increase the difficulty of control. Therefore, there is an urgent need for a long-lasting, green, efficient and precise control drug.

[0004] Chlorantraniliprole (CAP) is a diamide insecticide developed by DuPont. Studies have shown that the ryanodine receptor (RyR) is a Ca2+ receptor in the endoplasmic reticulum of insect muscle cells. 2+ Chlorantraniliprole acts on ryanodine receptors to release Ca 2+ The channel is open for a long time, resulting in the cytoplasmic Ca 2+Horizontal out-of-control rise eventually causes insect paralysis and death. Chlorantraniliprole is effective against various pests such as Lepidoptera, Coleoptera, Diptera, and Hemiptera. In paddy field production, chlorantraniliprole is mainly used to control Lepidoptera pests of rice (such as Chilo suppressalis, Tryporyza incertulas, Cnaphalocrocis medinalis) and Coleoptera pests (such as Lissorhoptrus oryzophilus, Leptinotarsa decemlineata). However, the overuse of chlorantraniliprole in the field has led to most pests developing high resistance to it, which poses a huge challenge to integrated pest management. In addition, the long-term use of chlorantraniliprole may also have negative impacts on non-target organisms, including natural enemies and aquatic organisms, and thus have a more extensive impact on the ecosystem.

[0005] Imidacloprid (IMI) is a broad-spectrum and highly effective nitro-substituted methylene systemic insecticide, belonging to the neonicotinoid insecticides of the chloronicotinyl class. It is characterized by low toxicity, low residue, and pests are not easily resistant to it, and has multiple effects such as contact toxicity, stomach toxicity, and systemic absorption. After pests come into contact with imidacloprid, the central nervous signal conduction is blocked, resulting in paralysis and death. Imidacloprid has good control effects on pests such as Cerambycidae, Scolytidae, Buprestidae, and Lissorhoptrus oryzophilus. However, due to its overuse in farmland, many rice pests have developed serious resistance. On March 9, 2023, the National Agricultural Technology Extension and Service Center issued a notice, suggesting that the main rice-producing areas should stop using imidacloprid.

[0006] In recent years, the progress of nanotechnology has provided innovative ways for pesticide controlled release. In particular, mesoporous silica nanoparticles (MSN) have become effective controlled release carriers due to their high loading efficiency, large specific surface area, and good biocompatibility. However, unmodified MSN cannot effectively control the release time and location of drugs. Through chemical modification, MSN can respond to environmental stimuli such as temperature, enzyme activity, pH value, and glutathione (GSH), thus achieving precise drug release. The introduction of carboxymethyl chitosan (CMCS) further improves its solubility, pH sensitivity, biocompatibility, and biodegradability, enhancing its environmental friendliness and application potential. In this study, the combination of mesoporous silica and carboxymethyl chitosan was used to chemically modify MSN through silane coupling agents and ionic binding to match the acidic microenvironment rich in glutathione in the midgut of the rice water weevil and the environmental response of pesticides. Through characterization and analysis such as SEM, TEM, FTIR, XPS, BET, TGA, and in vitro drug release experiments, it was proved that the prepared MSN-SS-CMCS nanocarrier successfully achieved dual responsiveness to pH and redox, and could adjust the drug release rate according to environmental changes. The results of bioactivity determination and safety evaluation showed that the MSN-SS-CMCS nano-pesticide controlled release system not only had an effective control effect on the rice water weevil, but also showed good environmental friendliness and sustainability, indicating broad application prospects. Summary of the Invention

[0007] The purpose of the present invention is to provide the application of a dual-controlled release nanocarrier loaded with chlorantraniliprole and imidacloprid in enhancing the control effect on the rice water weevil.

[0008] The dual-controlled release nanocarrier loaded with chlorantraniliprole and imidacloprid of the present invention is prepared by the following method:

[0009] Dissolve cetyltrimethylammonium bromide in water, adjust the pH value to alkaline, then add tetraethyl orthosilicate and (3-mercaptopropyl)trimethoxysilane for reaction. After the reaction is completed, wash with methanol and deionized water in sequence, and freeze-dry to obtain a dry powder. The dry powder is redispersed in a mixed solution of methanol and hydrochloric acid, and the CTAB template is removed by reflux reaction under a N2 atmosphere, and then obtained MSN-SH after centrifugal washing and drying;

[0010] MSN-SH reacts with 2,2′-dithiopyridine under a N2 atmosphere and in an acidic environment. The centrifuged precipitate is washed with methanol and deionized water to obtain MSN-SS-Py. MSN-SS-Py is dispersed in DMF containing acetic acid, and then 3-mercaptopropionic acid is added for reaction. The product is washed with methanol and deionized water to obtain MSN-SS-COOH;

[0011] MSN-SS-COOH was mixed and dispersed with 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide in PBS for reaction to activate the hydroxyl groups, and then carboxymethyl chitosan was added for further reaction. After centrifugation, washing and vacuum drying, MSN-SS-CMCS was obtained.

[0012] Chlorantraniliprole or imidacloprid was dissolved in absolute ethanol, and then MSN-SS-CMCS was added for reaction. Then, centrifugation was carried out, and the precipitate was washed with deionized water and absolute ethanol and vacuum dried to obtain CAP@MSN-SS-COOH. CAP@MSN-SS-COOH was mixed and dispersed with 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide in PBS for reaction to activate the hydroxyl groups, and then carboxymethyl chitosan was added for further reaction. After centrifugation, washing and vacuum drying, CAP@MSN-SS-CMCS or IMI@MSN-SS-CMCS was obtained.

[0013] Preferably, the pH environment in the body of the rice water weevil and the abundant glutathione are used to promote the application of the double-controlled release nano-carrier loaded with chlorantraniliprole and imidacloprid in the control of the rice water weevil.

[0014] The present invention also provides a double-controlled release nano-carrier loaded with chlorantraniliprole and imidacloprid prepared by the above preparation method.

[0015] The nano-pesticides CAP@MSN-SS-CMCS and IMI@MSN-SS-CMCS of the present invention can continuously and effectively control the rice water weevil and reduce the damage of the rice water weevil. Using the modified nano-pesticides can effectively reduce the usage amount of the original drug and reduce the pollution and toxicity of the pesticide to the environment. The nano-pesticides CAP@MSN-SS-CMCS and IMI@MSN-SS-CMCS are harmless to the growth of rice, and there are no phytotoxicity phenomena such as leaf rolling, leaf burning, and chlorosis. They have good safety for zebrafish, and the toxicity of the nano-pesticides is much less than that of the original drug. Description of the Drawings

[0016] Figure 1 It is the scanning and transmission electron microscope and particle size distribution diagram of MSN-SH, MSN-SS-COOH, and MSN-SS-CMCS.

[0017] Figure 2 It is the infrared spectrogram of MSN-SH, MSN-SS-COOH, MSN-SS-CMCS, and CMCS.

[0018] Figure 3 It is the X-ray photoelectron spectroscopy spectrum of MSN-SH, MSN-SS-COOH, and MSN-SS-CMCS.

[0019] Figure 4It is the BET-N2 adsorption-desorption isotherm curve and pore size distribution diagram.

[0020] Figure 5 It is the thermogravimetric analysis curve of MSN-SH, MSN-SS-COOH and MSN-SS-CMCS.

[0021] Figure 6 It is the standard curve of CAP at pH = 5.5, 6.5 and 7.4.

[0022] Figure 7 It is the standard curve of IMI at pH = 5.5, 6.5 and 7.4.

[0023] Figure 8 It is the static contact angle of CAP and IMI.

[0024] Figure 9 It is the adhesion performance detection of CAP and IMI.

[0025] Figure 10 It is the sustained release curve of CAP / IMI@MSN-SS-CMCS in different pH and GSH environments.

[0026] Figure 11 It is the corrected mortality rate of the modified nanocarrier MSN-SS-CMCS, the ordinary nanocarrier MSN encapsulated with CAP and IMI at different concentrations for 96 h against Lissorhoptrus oryzophilus Kuschel. Note: Each data is the average value obtained after 3 repetitions. Different lowercase letters in the figure represent that there is a significant difference in the mortality rate of Lissorhoptrus oryzophilus Kuschel after loading drugs with MSN and MSN-SS-CMCS under the same concentration treatment by t-test.

[0027] Figure 12 It is the effect of MSN-SS-CMCS on the germination rate, root length and chlorophyll value (SPAD) of rice seeds.

[0028] Figure 13 It is the median lethal concentration and fitting curve of different concentrations of CAP@MSN-SS-CMCS, CAP and IMI@MSN-SS-CMCS, IMI against zebrafish. Specific implementation mode

[0029] The following examples are further descriptions of the present invention, rather than limitations on the present invention.

[0030] Example 1:

[0031] I. Materials and methods

[0032] 1.1 Synthesis method of MSN-SS-CMCS

[0033] 1.1.1 Synthesis of MSN and MSN-SH

[0034] 1.1.1.1 Synthesis of MSN

[0035] Add 500 mg of CTAB to 240 mL of deionized water, heat to 80 °C and stir to dissolve. Add 3.5 mL of 1.00 mol / L NaOH, and dropwise add 2.0 mL of TEOS at a rate of 0.2 mL per minute while continuously stirring. After stirring for 3 hours, centrifuge the precipitate by freezing and wash it three times with methanol and deionized water. Vacuum freeze-dry to obtain a powdery product. Subsequently, disperse the powder in 80 mL of methanol and 4.5 mL of 1.00 mol / L hydrochloric acid, and reflux at 70 °C for 24 hours under a N2 atmosphere to remove the CTAB template. After centrifugation, washing, and drying, pure MSN is obtained.

[0036] 1.1.1.2 Synthesis of MSN-SH

[0037] Add 500 mg of CTAB (cetyltrimethylammonium bromide) to 240 mL of deionized water, and heat the mixture to 80 °C and continuously stir to ensure that CTAB is fully dissolved and uniformly dispersed. Subsequently, add 3.5 mL of 1.00 mol / L NaOH to the solution to adjust the pH to approximately 11. Under continuous stirring, slowly dropwise add 2.0 mL of TEOS (tetraethyl orthosilicate) at a rate of 0.2 mL per minute, and then add 0.5 mL of MPTMS ((3-mercaptopropyl)trimethoxysilane) at the same rate to ensure uniform mixing and introduction of functional groups. After the addition of MPTMS is completed, stir for another 3 h, centrifuge the precipitate using a refrigerated centrifuge (4 °C, 9000 r / min, 10 min), and perform three vortex washes using an equal volume mixture of methanol and deionized water, with each wash lasting 5 minutes to thoroughly remove all unreacted substances. After washing, vacuum freeze-dry the product to remove the solvent and obtain a dry powdery product.

[0038] Redisperse the dry powder in a mixed solution of 80 mL of methanol and 4.5 mL of 1.00 mol / L hydrochloric acid, and reflux at 70 °C for 24 h under a N2 atmosphere to completely remove the CTAB template through an acid-catalyzed hydrolysis reaction. After centrifugation, washing, and drying of the treated product, pure MSN-SH is obtained.

[0039] 1.1.2 Synthesis of MSN-SS-COOH

[0040] The pH value of the solution was adjusted to 2.0 by adding 1100 μL of 1.00 M HCl to 100 mL of deionized water. Under a N2 atmosphere, 500 mg of MSN-SH and 220 mg of 2,2′-dipyridyl disulfide were added to this solution, and it was stirred at room temperature for 24 h. After the reaction was completed, the precipitate product was separated by freeze centrifugation and washed repeatedly with methanol and deionized water to obtain MSN-SS-Py.

[0041] To further synthesize MSN-SS-COOH, 200 mg of MSN-SS-Py was dispersed in 15 mL of DMF containing 1.2 mL of acetic acid, and then 40 μL of 3-mercaptopropionic acid was added. After reacting at 40 °C for 24 h, MSN-SS-COOH nanoparticles were obtained, which were washed repeatedly with methanol and deionized water and stored in a refrigerator at 4 °C after drying.

[0042] 1.1.3 Synthesis of MSN-SS-CMCS

[0043] 100 mg of MSN-SS-COOH, 100 mg of EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide), and 80 mg of NHS (N-hydroxysuccinimide) were fully mixed and dispersed with PBS buffer, and the whole system was ultrasonically treated for 30 min to activate the carboxyl group. Then, 100 mg of carboxymethyl chitosan was added to 150 mL of 10% acetic acid aqueous solution, and the carboxymethyl chitosan was completely dissolved by shaking in a constant temperature shaker. The prepared carboxymethyl chitosan solution was added to MSN-SS-COOH, and the whole system was reacted at room temperature for 24 h. It was centrifuged at 9000 r / min for 10 min, washed several times, and vacuum dried for 24 h to obtain MSN-SS-CMCS.

[0044] 1.1.4 Synthesis of CAP@MSN-SS-CMCS and IMI@MSN-SS-CMCS

[0045] Using absolute ethanol as a solvent, a 2 mg·mL -1 solution of chlorantraniliprole (CAP) was prepared. 40 mg of MSN-SS-COOH was injected into a sample bottle, and 20 mL of a 2 mg·mL -1 CAP solution was added, and it was oscillated and adsorbed in a constant temperature water bath at room temperature for 24 h; it was centrifuged at 9000 r·min -1 for 10 min, washed several times with deionized water and absolute ethanol to remove excess pesticide residues, and vacuum dried for 24 h to obtain CAP@MSN-SS-COOH, and then CAP@MSN-SS-CMCS was obtained according to the steps in 1.1.3. Imidacloprid (IMI) was carried out according to the same steps to obtain IMI@MSN-SS-CMCS.

[0046] 1.1.5 Synthesis of CAP@MSN

[0047] Weigh 100 mg of MSN, and then add it to a 50 mL absolute ethanol solution containing 100 mg of CAP. Stir for 24 h. Centrifuge at 9000 r / min for 10 min, wash with deionized water and absolute ethanol several times to remove excess pesticide residues, and dry in vacuum for 24 h to obtain CAP@MSN. IMI is carried out according to the same procedure.

[0048] 1.1.6 The pH value in the body of the rice water weevil is acidic, ranging from 6 to 6.5

[0049] Grind the rice water weevil with an appropriate amount of PBS buffer solution with pH = 7.0, and use a pH test paper to detect. The measured result shows that the pH is in the range of 6.0 - 6.5.

[0050] 1.1.7 The body of the rice water weevil is rich in glutathione

[0051] According to the detection kit for the content of reduced glutathione (GSH), based on the concentration (x, μg / mL) and absorbance (ΔA standard, y) of the standard tubes, establish a standard curve. According to the standard curve, substitute ΔA (y, ΔA) into the formula to calculate the sample concentration (x, μg / mL). The standard curve equation of glutathione is Y = 0.0026X - 0.00295.

[0052] Take 0.0025 g of the rice water weevil, add 1 mL of reagent one for homogenization and grinding. After taking the supernatant, operate according to the measurement steps of the kit. The calculated ΔA = A measurement - A blank = 0.0338 is obtained by measuring with a 96-well plate. For the standard curve y = 0.0026x - 0.00295, x = 13.65. Calculated according to the sample mass: GSH content (μg / g mass) = x ÷ W = 5460 μg / g mass.

[0053] Glutathione exists widely in animals and plants and plays an important role in living organisms. Its content is very high in baker's yeast, wheat germ and animal liver, reaching 1 - 10 mg / g. In human blood, it contains 0.26 - 0.34 mg / g, in chicken blood it contains 0.58 - 0.73 mg / g, and in pig blood it contains 0.01 - 0.15 mg / g. By comparison with the above, the glutathione content in the body of the rice water weevil is 5.46 mg / g, which is relatively high.

[0054] 1.2 Characterization of nano-carrier materials

[0055] 1.2.1 Scanning electron microscopy and transmission electron microscopy detection

[0056] The morphology and structure of the nanoparticles were observed using a scanning electron microscope (SEM, Zeiss Gemini Sigma 300, Nova450; FEI-F50) and a transmission electron microscope (TEM, FEI-Talos F2000S). The particle size of the nanomaterial samples in the SEM images was measured using ImageJ software.

[0057] 1.2.2 Fourier transform infrared spectroscopy detection

[0058] The potassium bromide tablet method was used to analyze the changes in the functional groups of the carrier materials MSN-SH, MSN-SS-COOH, and MSN-SS-CMCS using a Fourier transform infrared spectrometer (FTIR, IRAffinity-1s). The spectral range was 400 - 4000 cm -1 .

[0059] 1.2.3 X-ray photoelectron spectroscopy detection

[0060] An X-ray photoelectron spectrometer (XPS, Kratos Axis Supra TM) was used to detect the elements and atomic valence state compositions present in the nanomaterials MSN-SH, MSN-SS-COOH, and MSN-SS-CMCS, and the change in the sulfur content of the elements on the sample surface was monitored.

[0061] 1.2.4 N2 adsorption-desorption specific surface area detection

[0062] A fully automatic specific surface area and porosity analyzer (BET-N2, Micromeritics ASAP2460 Version 3.01) was used. N2 was used as the analysis gas and its saturation pressure was measured. By measuring the adsorption volume at different adsorption pressures, the nitrogen adsorption-desorption isotherm was obtained. Then, the specific surface area, pore size, and pore volume of the nanomaterial samples, as well as the pore size distribution, were calculated using the Barrett-Emmett-Teller (BET) equation and the Barett-Joyner-Halanda (BJH) equivalent cylinder model.

[0063] 1.2.5 Thermogravimetric analysis test

[0064] Thermogravimetric detection was carried out using a thermogravimetric analyzer (TGA, Netzsch TG209) to investigate the modification rate and thermal stability of the carrier. The entire detection was carried out in an N2 environment, and the heating rate and temperature range were set to 10℃ / min and 25 - 800℃, respectively.

[0065] 1.3 Nanopesticide performance evaluation

[0066] 1.3.1 Plotting of the standard curve

[0067] The absorbance and concentration of two kinds of pesticide technicals were detected by using an ultraviolet spectrophotometer, and a standard curve was established to prepare for the subsequent detection of the drug loading rate, encapsulation rate and in vitro sustained release performance of the nanocarrier samples. Taking CAP as an example, the specific steps are as follows:

[0068] Weigh 4 mg of CAP, dissolve it in PBS buffer solutions with different pH values (pH = 5.5 / 6.5 / 7.4) to a volume of 10 mL to prepare a stock solution with a concentration of 400 mg / L, and store it in the dark at 4 °C. Then, the stock solution was serially diluted to prepare standard solutions with concentrations of 200 mg / L, 100 mg / L, 50 mg / L, 25 mg / mL, and 12.5 mg / L. Using PBS buffer as a control, the absorbance at 245 nm was measured with an ultraviolet spectrophotometer, and the CAP standard curve was plotted with OriginPro 2018. The standard curve was plotted with the concentration (c, mg / L) as the abscissa and the absorbance (A) as the ordinate, and the linear regression equation was obtained by fitting. The method for plotting the standard curve of IMI is the same as the above steps, and the detection wavelength is 270 nm.

[0069] 1.3.2 Determination of drug loading rate and encapsulation rate

[0070] Using absolute ethanol as a solvent, prepare a 2 mg / mL CAP solution. Inject 40 mg of MSN-SS-COOH into a sample bottle, add 20 mL of the 2 mg / mL CAP solution, and oscillate and adsorb it in a constant temperature water bath at room temperature for 24 h; centrifuge at 9000 r·min -1 Centrifuge for 10 min, and use an ultraviolet spectrophotometer to measure the concentration of unadsorbed CAP in the supernatant. IMI was also detected according to the above steps, and the drug loading rate (%) and encapsulation rate (%) were calculated according to the following formulas.

[0071] [[ID=!

[0072] Note: In the formula, m0 is the added mass of CAP, g; m1 is the remaining mass of CAP in the solution, g; m2 is the mass of MSN-SS-COOH after loading CAP or IMI, g.

[0073] 1.3.3 Determination of wetting property and adhesion property

[0074] Using a contact angle measuring instrument ( DSA100) Evaluate the wetting performance of the samples. Determine the wetting performance of CAP / IMI@MSN-SS-CMCS on rice leaves. Fix the rice leaf firmly on a glass slide, and then drop 3 μL of CAP / IMI@MSN-SS-CMCS suspension on the surface of the rice leaf. Each solution was repeated three times at different parts of the leaf, and the change in the contact angle was recorded. Operate on CAP / IMI@MSN-SS-CMCS, H2O, CAP@MSN, IMI@MSN, CAP, and IMI according to the above steps.

[0075] Weigh 5 mg each of CAP / IMI@MSN-SS-CMCS and mix them evenly with water to prepare an aqueous solution with a concentration of 100 mg / L. Take 20 mL as the solution required for the experiment. First, use a UV-visible spectrophotometer to detect the absorbance A before spraying the drug. Secondly, take 10 mL of the drug solution and spray it on fresh, clean rice leaves of basically the same size. After natural drying, soak the leaves in 10 mL of deionized water for 5 min, and use a UV-visible spectrophotometer to measure the absorbance B of the pesticide in the water. Finally, calculate the residual rate according to the formula.

[0076]

[0077] In the formula: M A is the mass of CAP / IMI at the start of spraying; M B is the mass of CAP / IMI remaining on the leaf after washing with water.

[0078] 1.3.4 Effects of PH and GSH on the in vitro drug release performance

[0079] Weigh 5.0 mg of CAP@MSN-SS-CMCS respectively and add them into dialysis bags (with a molecular weight cut-off of 8000 - 14000 Da) containing 3.0 mL of phosphate buffer solution (PBS, pH = 5.5 / 6.5 / 7.4). After placing the dialysis bags in centrifuge tubes, immediately add 30.0 mL of PBS (pH = 5.5 / 6.5 / 7.4) solution to the centrifuge tubes. Finally, place the centrifuge tubes containing CAP@MSN-SS-CMCS on a shaker and keep them oscillating at a constant temperature of 200 r / min in the dark for 96 h. Take out 3 mL of the release medium at specified times, measure the absorbance of CAP with a UV-visible spectrophotometer, and at the same time add 3.0 mL of fresh release medium to calculate the cumulative drug release percentage (CRP) of each sample.

[0080] Accurately weigh 5 mg of CAP@MSN-SS-CMCS, add it into a dialysis bag with a molecular weight cut-off of 8000 - 14000 Da, and then add 3 mL of PBS buffer with different GSH contents (0 mM, 4 mM, and 10 mM). Place the dialysis bag into 30 mL of phosphate buffer solution with the corresponding GSH content, set the temperature at 30 °C, and withdraw 3 mL of the sustained-release solution at fixed intervals for measurement. At the same time, add 3 mL of phosphate sustained-release solution with the corresponding GSH value, and detect the concentration of the pesticide in the release solution to calculate the cumulative release rate (CRP) of the drug for each sample. IMI@MSN-SS-CMCS is tested for pH and GSH sustained-release performance according to the above steps.

[0081]

[0082] Note: CRP: Cumulative release rate of the drug; Ve: Replacement volume of PBS; V0: Total volume of the release medium; C i : Concentration of the release solution at the i-th replacement sampling; M drug : Total mass of the drug carried by the nanoparticles; n: Number of times of replacing PBS.

[0083] 1.4 Toxicity determination and field application of nano-pesticides against Lissorhoptrus oryzophilus Kuschel

[0084] 1.4.1 Toxicity determination of nano-pesticides against Lissorhoptrus oryzophilus Kuschel

[0085] To determine the insecticidal activities of CAP@MSN-SS-CMCS nanoparticles and CAP@MSN against Lissorhoptrus oryzophilus Kuschel, the materials required to prepare CAP@MSN solution and CAP@MSN-SS-CMCS solution were taken to Wuhua County, Meizhou City, and prepared on-site on the day of the experiment. According to the results of the preliminary experiment, CAP@MSN-SS-CMCS and CAP@MSN were dispersed in water at five different concentrations of (800, 400, 200, 100, 50 mg / L) and (1600, 800, 400, 200, 100 mg / L), respectively. The rice water weevils starved for 24 h were then dispersed in petri dishes, with 10 rice water weevils and one rice seedling in each petri dish, and the rice seedlings were changed every 24 h. The rice water weevils were immersed in the solutions with different concentration treatments for 20 s using the dipping method. The control group was treated with MSN-SS-CMCS and water. At 12 h, 24 h, 48 h, and 96 h, the rice water weevils were gently touched with a brush. If the body curled up or it could not move normally for 15 - 20 s, it was regarded as dead, and the survival rate of the adult rice water weevils was recorded. Each treatment was repeated 3 times. Finally, the corrected mortality rate of the rice water weevils after 96 h was determined and its LC 50 50. IMI@MSN-SS-CMCS is carried out according to the above steps.

[0086] 1.4.2 Field application of nano-pesticides

[0087] 1.4.2.1 General situation of the test site

[0088] The test was carried out in 2024 in a paddy field of a farmer in Nanzhong Village, Longcun Town, Wuhua County, Meizhou City, Guangdong Province. The test site was flat with medium fertility, and surrounded by contiguous paddy fields.

[0089] 1.4.2.2 Test agents

[0090] Table 1 Design of treatments for field efficacy test of rice water weevil

[0091]

[0092] 1.4.2.2 Test methods

[0093] The test was designed by randomized block design, dividing into 15 plots with an area of about 10 m 2 each. Each plot was marked clearly for distinction. It included 4 agent treatments and 1 clear water control (the agent name and dosage design are shown in Table 3.1 specifically), and each treatment was repeated 3 times. To avoid edge effects and reduce test errors, treatment numbers 1 - 4 were the first - round treatments. Electric sprayers were used for spraying on August 9, and the spraying amount is shown in Table 1.

[0094] 1.4.2.3 Efficacy investigation

[0095] A total of 3 investigations were carried out. The population base number of rice water weevils was investigated on the day before spraying (August 8), and the efficacy was investigated 1 day (August 10) and 7 days (August 16) after spraying, counting the number of live rice water weevils and the leaf damage situation. The investigation method was the parallel jumping sampling method. In the paddy field, every several rows were skipped to take one row (or several rows) to investigate and record the number of adult rice water weevils and the number of damaged rice leaves. 10 clusters of rice were investigated in each plot, and the growth situation of rice was observed simultaneously. The reduction rate of insect population, corrected control efficacy, damage rate of rice leaves, and control effect were calculated and statistically analyzed. After converting the percentage value of the corrected control efficacy by the arcsine square root, using the DPS 7.05 software platform, the data were analyzed by Duncan's new multiple range method for variance analysis and multiple comparisons to analyze the significant differences among the results.

[0096]

[0097] 1.5 Safety evaluation of nano - pesticides

[0098] 1.5.1 Influence of MNS - SS - CMCS on the growth of rice and seeds

[0099] Dilute the MSN-SS-CMCS nanocarriers with water to different concentrations (0 (CK), 100, 200, 400, 800, 1600 mg / L). Then, coat filter paper and 20 seeds in each petri dish, soak the rice seeds in the MSN-SS-CMCS solution for 72 h. There are 3 replicates for each treatment. The blank control is deionized water without MSN-SS-CMCS. Check the seed germination rate after 72 h. Subsequently, hydroponically culture the rice for 2 weeks with different concentrations of the MSN-SS-CMCS solution and deionized water, and then measure the root length of the rice. Similarly, spray the rice seedlings with suspensions of different concentrations of the MSN-SS-CMCS nanocarriers. After 8 weeks, use a chlorophyll meter to measure the chlorophyll content (SPAD) of each treatment.

[0100] 1.5.2 Acute Toxicity Test of Nano-Pesticides on Adult Zebrafish

[0101] The type of zebrafish is wild-type zebrafish reared in the laboratory, with a body length of 2.04 ± 1.0 cm, healthy and disease-free. Before the formal experiment, the experimental zebrafish are pre-cultured for 7 d under the same environmental conditions as during the experiment. The mortality rate of zebrafish during the pre-culture period is <5%. Feed the zebrafish once a day during the pre-culture, promptly clean the debris in the fish tank to ensure the safety of the living environment of the zebrafish, maintain 12 h of daily light, and stop feeding 24 h before the formal experiment.

[0102] According to the preliminary experiment, the zebrafish stop feeding 24 h before the experiment. According to the preliminary experiment, 10 zebrafish are respectively exposed to the CAP / IMI@MSN-SS-CMCS water suspension with concentrations of 1600, 800, 400, 200, 100 mg / L. The treatment concentrations of the CAP and IMI technical materials are 150, 120, 90, 60, 30 mg / L. At the same time, set up the MSN-SS-CMCS exposure group without CAP and IMI and the blank control group at the corresponding concentrations, with each treatment repeated three times. Observe and record the poisoning symptoms and the number of dead fish of the experimental fish 12 h after the start of the experiment, and record the death conditions of the zebrafish at 12 h, 24 h, 48 h, 72 h, and 96 h. If there is no response when gently touching the tail of the fish with a glass rod, the fish is considered dead. The dead zebrafish are quickly removed to reduce the pollution of the culture water by the dead fish. Finally, measure the corrected mortality rate and LC of the 96-h zebrafish for CAP, IMI, CAP@MSN-SS-CMCS, and IMI@MSN-SS-CMCS 50 。

[0103] 2. Results Analysis

[0104] 2.1 Scanning Electron Microscope and Transmission Electron Microscope Analyses of Nanocarriers

[0105] 2.1.1 Morphological Structure Analysis

[0106] The observation results of the scanning electron microscope showed that the prepared MSN-SS-CMCS particles were generally spherical, with uniform particle size and rough surface structure, and had a dense mesoporous structure (see Figure 1 ). Further transmission electron microscope observation results showed that the particle size of MSN-SS-CMCS was relatively uniform, the mesoporous structure was clear, and the inner cavity space ( Figure 1 ). The average particle sizes of MSN-SH, MSN-SS-COOH, and MSN-SS-CMCS nanoparticles were 116 nm and 143 nm respectively. Due to the further modification with disulfide bonds and carboxyl groups, the particle size increased further. Finally, through the encapsulation of carboxymethyl chitosan, the diameter of the nanoparticles could reach 168 nm ( Figure 1 ).

[0107] 2.1.2 Other characterization structure analysis

[0108] 2.1.2.1 Infrared spectroscopy analysis results

[0109] It can be seen from Figure 2 that on the infrared spectrum of MSN-SH, the stretching and bending vibration absorption peaks of Si-OH are at 3392 cm -1 and 960 cm -1 , the stretching vibration absorption peaks of C-H are at 2926 cm -1 and 2858 cm -1 , and the vibration absorption peaks of Si-O-Si are at 1051 and 794 cm -1 , indicating the successful synthesis of mesoporous silica. In addition, the characteristic absorption peak of -SH is at 2565 cm -1 , indicating the successful modification of mercapto groups. It can be seen from the infrared spectrum of MSH-SS-COOH that the stretching and bending vibration absorption peaks of Si-OH are at 3425 cm -1 and 960 cm -1 , and the stretching vibration absorption peak of C-H is at 2928 cm -1 . Due to the introduction of disulfide bonds and carboxyl groups, the mercapto characteristic peak disappears, and the stretching vibration absorption peak of C=O on the carboxyl group appears at 1627 cm -1 , and the vibration absorption peaks of Si-O-Si on the mesoporous silica are at 1074 and 796 cm -1 .

[0110] It can be seen from the infrared spectrum of CMCS that the stretching vibration absorption peaks of N-H and O-H are at 3411 cm -1 , the stretching vibration absorption peak of C-H is at 2914 cm -1 , the stretching vibration absorption peak of C=O on the carboxyl group appears at 1618 cm -1 , and the stretching vibration absorption peak of C=O on the carboxyl group appears at 1426 cm -1The absorption peak at this position is the bending vibration absorption peak of -CH2-, 1127 cm -1 The absorption peak at this position is the stretching vibration absorption peak of C-O-C, 1059 cm -1 The absorption peak at this position is the in-plane bending vibration absorption peak of C-H, 705 cm -1 The absorption peak at this position is the out-of-plane bending vibration absorption peak of C-H.

[0111] It can be seen from the infrared spectrum of MSH-SS-CMCS that at 3425 cm -1 and 959 cm -1 The absorption peaks at this position are the stretching and bending vibration absorption peaks of Si-OH. At the same time, at 3425 and 2930 cm -1 The absorption peaks at this position are the stretching vibration absorption peaks of C-H on MSH-SS-CMSS. 1657 cm -1 The absorption peak at this position is the stretching vibration of (amide I band) C=O, 1573 cm -1 The absorption peak at this position is the stretching vibration of (amide II band) N-H, 1413 cm -1 The absorption peak at this position is the stretching vibration of (amide III band) C-N, indicating the successful introduction of CMCS. 1077 and 799 cm -1 The absorption peaks at this position are the vibration absorption peaks of Si-O-Si on mesoporous silica.

[0112] 2.1.2.3 X-ray photoelectron spectroscopy (XPS) spectrum analysis

[0113] Figure 3 It can be seen from the C1s diagram of that there are mainly C-C (284.8 eV) and C-S (286.0 eV) bonds on MSH-SH. Among them, C-C mainly comes from the inevitable foreign contaminant carbon in the test, and the C-S bond comes from the grafting of -SH bond to MSN. Due to the introduction of COOH, a C=O bond appears at the 284.5 eV position in MSN-SS-COOH. Due to the modification of CMCS, a C-N bond (285.5 eV) appears on MSN-SS-CMCS. It can be seen from the S2p diagram that there is a C-S bond at 163.5 eV, and an R-SH bond (R refers to MSN) at 164.7 eV. After modification with 3-mercaptopropionic acid, the R-SH bond of MSN-SS-COOH disappears and is replaced by an S=S bond at 164.9 eV.

[0114] It can be seen from the XPS suvery diagram that there are obviously four elements, Si, O, C and S, on MSH-SH. The appearance of S proves the grafting of -SH to MSN. There is a peak of N1s on MSN-SS-CMCS, which is mainly due to the introduction of N element by the modification of CMCS. These results indicate the success of the modification of the nanocarrier.

[0115] 2.1.2.4 BET-N2 Adsorption and Desorption Results Analysis

[0116] To determine the specific surface area and pore structure of the nanomaterials, BET characterization was carried out on the synthesized nanomaterials MSN-SH, MSN-SS-COOH, and MSN-SS-CMCS. The N2 adsorption isotherms and pore size distributions of the nanomaterials are as Figure 4 shown. From the N2 adsorption isotherms, it can be seen that for different materials, the adsorption amount increases gently at low and medium pressure ratios, while there is a sharp increase in the adsorption amount at high pressure ratios (0.8 - 1.0), and an obvious adsorption hysteresis phenomenon also appears. This belongs to a typical Type V isotherm, indicating that micropores and mesopores exist in these nanomaterials. This phenomenon is particularly obvious in the MSN-SS-CMCS material, indicating that its pore structure is more abundant. The BET specific surface areas of MSN, MSN-SS-COOH, and MSN-SS-CMCS are 86.3769 m 2 / g, 37.8492 m 2 / g, and 120.1512 m 2 / g respectively, indicating that different synthesis methods have a significant impact on the specific surface area of the products. Among them, MSN wrapped with carboxymethyl chitosan has the largest specific surface area, which is beneficial to improving the performance of the nanomaterials.

[0117] The results of the pore size distribution further show that the pore diameters of the MSN-SH and MSN-SS-COOH samples are mainly concentrated between 20 - 30 nm, mainly being mesoporous structures, while the pore diameters of the MSN-SS-CMCS sample are distributed below 20 nm, indicating that it has a hierarchical porous structure of micropores and mesopores, which is beneficial to improving the activity of the material. And, from the distribution of their pore volumes, that of MSN-SS-CMCS is 0.4736 cm 3 / g, much higher than that of MSN-SS-COOH which is 0.1540 cm 3 / g, indicating that MSN wrapped with carboxymethyl chitosan has a more abundant pore structure. From the analysis of the specific surface area and pore structure, it can be concluded that the existence of the rich pore structure and the large specific surface area of MSN-SS-CMCS are beneficial to improving the reaction activity of the nano-pesticide.

[0118] Table 2 Specific Surface Area, Pore Volume, and Average Pore Diameter of MSN-SH, MSN-SS-COOH, and MSN-SS-CMCS

[0119]

[0120] 2.1.2.5 Thermogravimetric Analysis Results

[0121] The thermal stabilities of MSN-SH, MSN-SS-COOH, and MSN-SS-CMCS were analyzed by a thermogravimetric analyzer as shown in Figure 5 . It can be seen from the TG-DTG curves that mass losses occurred for all three within the temperature range of 25 - 150 °C, which was mainly caused by the evaporation of adsorbed water on their surfaces. When the temperature rose to 150 °C, all the above moisture substances volatilized completely, and a small weight loss peak also confirmed this. Continuing to increase the temperature caused further weight loss for the three, and the continuous weight loss within the range of 150 - 500 °C was due to the decomposition of the substances grafted on their surfaces. The relatively large decomposition rate of MSN-SH in the DTG curve may be directly related to the decomposition of the mercapto groups connected to its surface. The double peaks that appeared in the DTG curve of MSN-SS-COOH were due to the decomposition of the disulfide bonds and carboxyl groups on its surface at high temperatures. The significantly reduced maximum decomposition rate of MSN-COOH indicates that the disulfide bonds have a positive effect on thermal stability.

[0122] The maximum decomposition rate of MSN-SS-CMCS was significantly less than the previous two, which may be because CMCS is rich in carbon elements, and during the pyrolysis process, it promoted the formation of a dense carbon layer structure, reducing the thermal-triggered mobility of the molecular chains and thus having an inhibitory effect on thermal degradation. After 500 °C, the weight loss of the three was slow, which was because the solid coke and inert residues formed during the pyrolysis process were thermally stable. In addition, at 800 °C, the undissolved residues of the three were all above 58%, and MSN-SS-CMCS reached 71%. Therefore, MSN-SS-CMCS has relatively excellent thermal stability performance.

[0123] 2.2 Detection of the performance of nano-pesticides

[0124] 2.2.1 Standard curves and drug loading rates

[0125] The standard curves of CAP at pH = 5.5, 6.5, and 7.4 are as shown in Figure 6 . The standard curves of IMI at pH = 5.5, 6.5, and 7.4 are as shown in Figure 7 .

[0126] Through the standard curves, drug loading rate and encapsulation efficiency experiments were carried out, and the drug loading rate of CAP@MSN-SS-CMCS was measured to be 38.76%, and the encapsulation efficiency was 63.31%; the drug loading rate of IMI@MSN-SS-CMCS was 48.21%, and the encapsulation efficiency was 93.10%. The drug loading situation and drug encapsulation situation of the nano-carriers were good.

[0127] Table 3 Drug loading rates and encapsulation efficiencies of nano-carriers

[0128]

[0129] 2.2.2 Wettability and adhesion performance

[0130] In Figure 8 , the contact angle of water on rice leaves is 125.4°, which is greater than 90°, indicating that the surface of rice leaves is hydrophobic. The contact angle of the CAP aqueous solution with the leaves is 129°, and the CAP aqueous solution does not wet the rice leaves. The contact angles of MSN@CAP and CAP@MSN-SS-CMCS on rice leaves are 124.0° and 122.2°, respectively, both greater than 90°, indicating that the contact angles of the nanocarriers encapsulating CAP have decreased, but they do not wet the rice leaves.

[0131] The contact angle of the IMI aqueous solution with the leaves is 111.2°, indicating that the IMI aqueous solution does not wet the rice leaves. The contact angles of IMI@MSN and IMI@MSN-SS-CMCS on rice leaves are 115.8° and 112.6, respectively. After the MSN-SS-CMCS carrier encapsulates IMI, the contact angle increases slightly, with little change.

[0132] The results of the adhesion performance test show that ( Figure 9 ), the remaining rates of using MSN-SS-CMCS to load CAP and IMI are increased from the original 7.46% and 5.76% to 34.76% and 17.86%, respectively, and the adhesion performance is significantly improved, indicating that the MSN-SS-CMCS carrier can greatly improve the adhesion performance of pesticides on rice leaves and reduce the drift of pesticides due to weather and environmental factors.

[0133] 2.2.3 pH and GSH Sustained Release Performance

[0134] Figure 10 is the sustained release curve of CAP / IMI@MSN-SS-CMCS under different pH and GSH environments

[0135] By evaluating the cumulative release rate of nanoparticles in different pH environments, it is found that the pH value and GSH content play crucial roles in the release of pesticides. In the initial stage of drug release, the pore volume of the nano-silica particles is large. Due to the gradual decrease of the drug concentration difference, with the extension of time, the cumulative release rate first gradually increases and then tends to be stable. When pH = 5.5, the highest cumulative release rate of CAP from CAP@MSN-SS-CMCS is about 16.70%. At the same time, the highest cumulative release rates at pH values of 6.8 and 7.4 do not exceed 8.9%. The cumulative release rates of IMI@MSN-SS-CMCS at different pH values in the first 96 hours of release are 6.40%, 3.83%, and 1.81%, respectively. The drug release rates of CAP and IMI under different pH environmental conditions are better than those of CAP. The above results indicate that CAP@MSN-SS-CMCS and IMI@MSN-SS-CMCS have good pH responsiveness.

[0136] It can be seen from Figure 10 that after treatment with different contents of GSH in CAP@MSN-SS-CMCS, the cumulative drug release rate at the initial stage of drug release changed little. However, after 8 h, the cumulative drug release rate of the treatment groups with 4 mM and 10 mM GSH increased, and the highest reached 72.45%. This is because at the initial stage of drug release, the disulfide bonds were not completely broken, and the groups contained in methyl chitosan were not reacted, resulting in a low drug release rate. However, with the extension of time, the disulfide bonds gradually broke, and the drug was gradually released. This indicates that the introduction of GSH can increase the drug release rate, and it also shows that the carrier has good redox performance. Without the addition of GSH, the release rate of CAP pesticide is even less than 10.45%. In contrast, after 4 mM and 10 mM GSH, the release rates of IMI@MSN-SS-CMCS reached 13.47% and 16.31% respectively. When the GSH concentration increased to 10 mM, the cumulative drug release rate was higher than that at 4 mM at the same time, indicating that a richer GSH is more conducive to drug release.

[0137] 2.3 Toxicity determination and field application of nano-pesticides against Lissorhoptrus oryzophilus Kuschel

[0138] 2.3.1 Toxicity determination of nano-pesticides against Lissorhoptrus oryzophilus Kuschel

[0139] As Figure 11 shown, through t-test, it was found that the insecticidal effects of pesticides encapsulated by modified nano-carriers, namely CAP@MSN-SS-CMCS and IMI@MSN-SS-CMCS, were significantly higher than those of ordinary nano-carriers, namely CAP@MSN and IMI@MSN, at the same concentration treatment. After 96 h, the highest corrected mortality rates of Lissorhoptrus oryzophilus Kuschel by the two nano-pesticides CAP@MSN-SS-CMCS and IMI@MSN-SS-CMCS were 82.96±5.13% and 82.59±6.51% respectively. After calculation and analysis by Graphpad Prism 8, the LC 50 of CAP@MSN-SS-CMCS was 187.0 mg / L, and the LC 50 of CAP@MSN was 718.8 mg / L. The efficacy of CAP loaded on the modified nano-carrier was 3.84 times higher than that of CAP loaded on ordinary MSN. The LC 50 of IMI@MSN-SS-CMCS was 101.4 mg / L, and the LC 50= 557.4 mg / L. The efficacy of the modified nano-carrier loaded with IMI is 5.50 times higher than that of the ordinary MSN loaded with IMI. It can be seen from the above results that the effect of reducing drug dosage and enhancing efficacy of the modified nano-carrier is very significant. Due to the permeability-enhancing, adhesiveness and stability of CMCS, the loaded drug can more easily penetrate the outer shell of the target pest, and then reach the effective insecticidal concentration through the dual environmental responsiveness to pH and GSH, improving the efficacy of pesticides while avoiding unnecessary drug waste.

[0140] 2.3.1 Field application of nano-pesticides

[0141] According to Table 4, 1 day after spraying, the population decline rate and corrected control effect of the CAP@MSN-SS-CMCS treatment group were the highest, which were 19.44% and 24.60% respectively. The population decline rate and corrected control effect of the CAP treatment group were the lowest, which were 10.98% and 16.78% respectively. 7 days after spraying, the population decline rate and corrected control effect of the CAP@MSN-SS-CMCS treatment group were the highest, which were 94.44% and 98.05% respectively; followed by the IMI@MSN-SS-CMCS treatment group, with the population decline rate and corrected control effect being 91.07% and 93.40% respectively; the population decline rate and corrected control effect of the IMI treatment group were the lowest, which were 77.59% and 75.31% respectively. By comparing each treatment group, it was found that the population decline rate of the modified nano-pesticide treatment group was higher than that of the original drug group, and the corrected control effect was significantly higher than that of the original drug treatment group.

[0142] Thus, it can be seen that the modified nano-pesticides can also continuously and effectively control Lissorhoptrus oryzophilus in the experimental field, reducing the damage of Lissorhoptrus oryzophilus. Using the modified nano-pesticides can effectively reduce the usage amount of the original drug, reducing the pollution and toxicity of pesticides to the environment. The experiment also paid attention to the growth of rice in the treatment group and the control group on each efficacy investigation day. There was no significant difference between the two visually, and there were no phytotoxicity phenomena such as leaf rolling, leaf burning, and chlorosis, indicating that the dosage of the above four groups of treatment agents was safe.

[0143] Table 4 Population decline rate and control effect of Lissorhoptrus oryzophilus before and after spraying

[0144]

[0145] Note: Each data is the average value obtained after 3 repetitions. The same marked letters after the data in the same column in the table indicate no significant difference, and different marked letters indicate significant difference. Among them, lowercase letters indicate a significant level (P < 0.05), and uppercase letters indicate a highly significant level (P < 0.01).

[0146] According to Table 5, 1 day before pesticide application, the damage rate of leaves was 25.47% - 30.97%, and the damage level was medium (++). 1 day after pesticide application, there was no obvious change in the damage rate of rice leaves. The control effect of CAP@MSN-SS-CMCS was better, at 15.33%; the control effect of IMI was relatively the worst, at 12.43%. 7 days after pesticide application, the damage rate of leaves was 14.35% - 15.62%, and the damage level dropped to slight (+). The control effects of CAP@MSN-SS-CMCS, CAP, IMI@MSN-SS-CMCS, and IMI on the leaves damaged by Lissorhoptrus oryzophilus Kuschel were 60.78%, 60.16%, 68.73%, and 57.14% respectively, and there was no significant difference among the four groups of treatments. By comparing the results 1 day before and 7 days after pesticide application, it was found that the damage rate of rice leaves decreased significantly. The main reason was that the sprayed pesticides inhibited the damage of Lissorhoptrus oryzophilus Kuschel, resulting in a reduction in the number of damaged leaves. During the experiment, the growth of rice in the treatment group and the control group was also observed on each day of efficacy investigation. There was no significant difference between the two visually, and there were no phytotoxicity phenomena such as leaf rolling, leaf burning, and chlorosis, indicating that the application doses of the pesticides in the above four groups were safe.

[0147] Table 5 Damage rate and control effect of Lissorhoptrus oryzophilus Kuschel on rice leaves after pesticide application

[0148]

[0149] Note: All data are the averages obtained after 3 repetitions. The same marked letters after the data in the same column in the table indicate no significant difference, and different marked letters indicate significant difference. Among them, lowercase letters represent the significant level (P < 0.05), and uppercase letters represent the extremely significant level (P < 0.01).

[0150] 2.4 Safety evaluation

[0151] 2.4.1 Rice crops

[0152] The determination of plant growth indicators sensitive to environmental changes can be used to evaluate the safety of the carrier MSN-SS-CMCS on rice growth. Figure 12Shows the effects of MSN-SS-CMS on the germination rate (A) and root length (B) of rice. After 72 h of treatment, in the water control group (0 mg / L), the germination rate of rice was 76.67%, and there was no significant difference compared with that of rice treated with different concentrations of MSN-SS-CMCS. After 2 weeks of treatment, the root length of the control group of rice was 4.87 cm, and the root length of the treated group of rice was in the range of 4.53 - 5.53 cm, with no significant difference. The SPAD values (an index reflecting chlorophyll content) of rice treated with different concentrations of MSN-SS-CMCS were in the range of 36.77 - 39.7, and the SPAD value of the control group of rice was 37.87, showing no significant statistical difference. These data results indicate that the MSN-SS-CMCS nanocarrier has no adverse effects on the growth and development of crops, confirming its good safety. It can be concluded that MSN-SS-CMCS can not only be used as an effective release system for plant-derived pesticides and chemical pesticides, achieving precise controlled release for pest control through stimulus response, but also is safe and harmless to the treated crops. This provides an important safety guarantee for the application of MSN-SS-CMCS, supporting its potential as an environmentally friendly and efficient pesticide release carrier in agricultural practice.

[0153] 2.4.2 Aquatic organism zebrafish

[0154] In the environmental risk assessment of pesticides in water, zebrafish is an important biological test material for acute toxicity tests of pesticides on fish. Figure 13 Shows the acute toxicity of CAP and CAP@MSN-SS-CMCS, as well as IMI and IMI@MSN-SS-CMCS to zebrafish under 96 h of exposure time. After 96 h, the LC 50 values of CAP and CAP@MSN-SS-CMCS were 110.69 mg / L and 1440 mg / L respectively, and the LC 50 values of IMI and IMI@MSN-SS-CMCS were 115.5 mg / L and 800.02 mg / L respectively. According to the acute toxicity classification of pesticides to fish, the LC 50 of CAP@MSN-SS-CMCS, CAP, IMI@MSN-SS-CMCS, and IMI to zebrafish at 96 h were all greater than 10 mg / L, indicating low toxicity (low toxicity: LC 50 > 10 mg / L). Comparing the LC 50 of the original drug and the nano-pesticide, we can find that the toxicity of the nano-pesticide is much lower than that of the original drug, with good safety.

Claims

1. A preparation method of a double-controlled release nano-carrier loaded with chlorantraniliprole and imidacloprid, characterized in that, It includes the following steps: Dissolve cetyltrimethylammonium bromide in water, adjust the pH value to alkaline, then add tetraethyl orthosilicate and (3-mercaptopropyl)trimethoxysilane for reaction. After the reaction is completed, wash with methanol and deionized water in sequence, and freeze-dry to obtain a dry powder. Redisperse the dry powder in a mixed solution of methanol and hydrochloric acid, reflux and react under a N2 atmosphere to remove the CTAB template, and then obtain MSN-SH after centrifugal washing and drying; React MSN-SH with 2,2′-dithiopyridine under a N2 atmosphere and in an acidic environment. Wash the centrifugally separated precipitate with methanol and deionized water to obtain MSN-SS-Py. Disperse MSN-SS-Py in DMF containing acetic acid, then add 3-mercaptopropionic acid for reaction, and wash the product with methanol and deionized water to obtain MSN-SS-COOH; Mix MSN-SS-COOH with 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, disperse them in PBS for reaction to activate the hydroxyl group, then add carboxymethyl chitosan for further reaction, and obtain MSN-SS-CMCS after centrifugal washing and vacuum drying; Chlorantraniliprole or imidacloprid is dissolved in absolute ethanol, then add MSN-SS-CMCS for reaction, then centrifuge, wash the precipitate with deionized water and absolute ethanol, and obtain CAP@MSN-SS-COOH after vacuum drying. Mix CAP@MSN-SS-COOH with 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, disperse them in PBS for reaction to activate the hydroxyl group, then add carboxymethyl chitosan for further reaction, and obtain CAP@MSN-SS-CMCS or IMI@MSN-SS-CMCS after centrifugal washing and vacuum drying.

2. A dual-controlled release nanocarrier loaded with chlorantraniliprole and imidacloprid prepared by the preparation method according to claim 1.

3. Application of the dual-controlled release nanocarrier loaded with chlorantraniliprole and imidacloprid according to claim 2 in improving the control effect of Lissorhoptrus oryzophilus Kuschel.

4. Application of the pH environment in the body of Lissorhoptrus oryzophilus Kuschel and abundant glutathione in promoting the application of the dual-controlled release nanocarrier loaded with chlorantraniliprole and imidacloprid according to claim 2 in controlling Lissorhoptrus oryzophilus Kuschel.

5. The application according to claim 4, characterized in that, The pH environment described is pH 6 - 6.5.

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