Active oxygen response nano-piperonyl butoxide synergist complex and application in synergistic high-efficiency cypermethrin control effect on aphids

Abstract

The present application relates to the technical field of pesticides, and aims to solve the problem of how to improve the insecticidal effect of high-efficiency cypermethrin on aphids resistant to the pesticide, and discloses an active oxygen responsive nano-piperonyl butoxide synergist compound and application thereof in synergizing the control effect of high-efficiency cypermethrin on aphids. The active oxygen responsive nano-piperonyl butoxide synergist compound is composed of PLGA-TK-PEG and piperonyl butoxide loaded therein. Compared with free piperonyl butoxide, the active oxygen responsive nano-piperonyl butoxide synergist has the following advantages: rapid-acting property is enhanced, stability is greatly improved, can be released in response to ROS, has super-high resistance reversal capability, and the synergistic effect with high-efficiency cypermethrin is up to 54.22, and the 7-day control effect on resistant aphids is increased from 61.38% of free PBO treatment to 84.06%. Moreover, the active oxygen responsive nano-piperonyl butoxide synergist can be targetedly delivered, the carrier is biodegradable, PBO is released on demand, and the use amount and environmental residue can be reduced.

Description

Technical Field

[0001] This invention relates to the field of pesticide technology, and specifically discloses a reactive oxygen species-responsive nano-piperidine butyl ether synergist complex and its application in enhancing the aphid control effect of cypermethrin. Background Technology

[0002] The long-term and extensive use of chemical pesticides has led to a continuous increase in pesticide resistance among pests, which has become a key issue in global agricultural production. High-efficiency cypermethrin, as a representative pyrethroid insecticide, is widely used to control wheat aphids (including the wheat aphid, wheat long-tubed aphid, and wheat two-forked aphid), but high levels of resistance (thousands of times higher) have emerged in the field, resulting in a significant decrease in the efficacy of conventional pesticides.

[0003] Piperidin (PBO) is a classic inhibitor of cytochrome P450 detoxification enzymes and can effectively reverse insect resistance to pyrethroids. However, PBO has several key drawbacks: 1) It has extremely poor water solubility and is difficult to disperse evenly in water-based systems.

[0004] 2) Uncontrollable release: Free PBO is metabolized in the insect body within 12–24 hours, resulting in a narrow window for synergistic effects and low utilization rate.

[0005] Existing technologies often involve directly combining free PBO with insecticides, which fails to address issues of stability, targeting, and long-lasting effects, making it difficult to meet the control requirements of highly resistant aphids.

[0006] Therefore, developing stable, long-lasting, and intelligently responsive nanodelivery systems to achieve targeted delivery and controlled release of PBO is of great significance for overcoming the bottleneck of pyrethroid drug resistance. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of existing PBO applications for aphids resistant to high-efficiency cypermethrin by providing a ROS-responsive nano-piperyl butyl ether synergist PLGA-TK-PEG-PBO. This synergist significantly improves the water solubility, stability, and bioavailability of PBO. PBO is intelligently released in a ROS-rich microenvironment generated by insecticide stress, providing long-term inhibition of P450 enzymes. This achieves long-term, highly synergistic control of aphids by combining PBO with high-efficiency cypermethrin, greatly enhancing the toxicity of high-efficiency cypermethrin to resistant aphids and providing a new technology for the green and efficient control of resistant aphids.

[0008] To achieve the above objectives, the present invention provides the following technical solution: The present invention first provides a reactive oxygen species responsive nano-piperyl butyl ether synergist complex, which is composed of PLGA-TK-PEG and its encapsulated piperin butyl ether (PBO, CAS No. 51-03-6).

[0009] In the above-mentioned composite, the PLGA-TK-PEG is a ROS (reactive oxygen species) responsive nanocarrier, which is composed of a hydrophilic PEG (polyethylene glycol) shell, a hydrophobic PLGA (polylactic acid-glycolic acid copolymer) core, and a ROS (reactive oxygen species) responsive switch TK (thioketal bond).

[0010] In the above complex, the piperonyl butyl ether is the active ingredient.

[0011] In the above-mentioned complex, the drug loading (the mass percentage of the encapsulated drug relative to the total mass of the drug-loaded nanoparticles) of the reactive oxygen species-responsive nanopiperyl butyl ether synergist complex is 8%-12%. In one embodiment of the present invention, the drug loading of the reactive oxygen species-responsive nanopiperyl butyl ether synergist complex is 10.6% (i.e., the mass ratio of PLGA-TK-PEG to piperin butyl ether is 8.48:1).

[0012] The present invention also provides a method for preparing the reactive oxygen species-responsive nano-piperyl butyl ether synergist complex, which is prepared by an emulsification-solvent evaporation method, namely, adding the PLGA-TK-PEG and the piperyl butyl ether to an organic solvent, adding a surfactant, emulsifying, then evaporating the organic solvent and collecting the precipitate to obtain the reactive oxygen species-responsive nano-piperyl butyl ether synergist complex.

[0013] In the above method, the organic solvent is at least one of dichloromethane and trichloromethane.

[0014] In the above method, the function of the surfactant is to reduce interfacial tension and prevent droplet coalescence, and a nonionic surfactant is used. The nonionic surfactant can be selected from any one of polyvinyl alcohol, polyoxyethylene sorbitan monooleate, and polyoxyethylene polyoxypropylene block copolymer.

[0015] In one embodiment of the present invention, the organic solvent is dichloromethane and the surfactant is polyvinyl alcohol (PVA).

[0016] The above method also includes a step of freeze-drying the precipitate.

[0017] In one embodiment of the present invention, the method specifically includes the following steps: 1) Weigh 50 mg of PLGA-TK-PEG and 2.65 mg of piperonyl butyl ether, and dissolve them in 1 mL of dichloromethane organic solvent; 2) Add 5 mL of 5% (v / v) polyvinyl alcohol aqueous solution and ultrasonically emulsify at 300 W for 10 min; 3) Stir at 500 rpm for 1 h at room temperature to evaporate the organic solvent; 4) Centrifuge at 4000 rpm for 5 min to remove large particles and collect the supernatant; 5) Centrifuge the supernatant at 13000 rpm for 5-10 min and collect the precipitate; 6) Wash the precipitate twice with water and freeze-dry to obtain the active oxygen responsive nano-piperidine butyl ether synergist complex.

[0018] The present invention also provides the application of the active oxygen-responsive nano-piperyl butyl ether synergist complex in enhancing the aphid control effect of cypermethrin.

[0019] In the above applications, the aphid can be the cereal constrictor aphid (Aphis graminifolia). Rhopalosiphum padi ) and / or wheat aphid ( Sitobion avenae ).

[0020] In the above applications, the aphids may be aphids resistant to high-efficiency cypermethrin.

[0021] In one embodiment of the present invention, the aphid is a lambda-trapping aphid resistant to highly efficient cypermethrin.

[0022] In the above applications, the mass ratio of the active oxygen-responsive nano-piperidine butyl ether synergist complex to the high-efficiency cypermethrin is 1.12:1.

[0023] In the above applications, the working concentration of the high-efficiency cypermethrin is 50 mg / L - 800 mg / L, the preferred working concentration is 200 mg / L - 400 mg / L, and the optimal working concentration is 223.7 mg / L.

[0024] The beneficial effects of this application are as follows: 1. Enhanced speed of action The PLGA-TK-PEG nanocarrier endows PBO with rapid penetration and immediate release properties, enabling rapid onset of action after application and solving the problems of delayed onset of action and gaps in initial control caused by free PBO.

[0025] 2. Stability has been greatly improved. PLGA-TK-PEG nanocarriers effectively protect PBO, prolong its duration of effectiveness, and solve the problem of poor water solubility.

[0026] 3. ROS Intelligent Response Release Precise drug release in pesticide-induced ROS microenvironment extends the synergistic window from several hours to over 48 hours.

[0027] 4. Extremely high resistance and reversal ability With a synergistic effect ratio of 54.22, it effectively overcomes the extremely high level of resistance (>1000 times) of aphids to high-efficiency cypermethrin, achieving efficient control of resistant populations.

[0028] 5. Targeted delivery and high utilization rate Nanoscale size facilitates penetration and endocytosis, increasing PBO accumulation in aphids and reducing non-target exposure.

[0029] 6. Environmentally friendly PLGA-TK-PEG is biodegradable, and PBO is released on demand, which can reduce dosage and environmental residues. Attached Figure Description

[0030] Figure 1 These are transmission electron microscopy (TEM) images of PLGA-TK-PEG and PLGA-TK-PEG-PBO from embodiments of the present invention. Figure 1 A is PLGA-TK-PEG. Figure 1 B is PLGA-TK-PEG-PBO.

[0031] Figure 2 The images show the particle size distribution, Zeta potential, and Fourier transform infrared spectra of PLGA-TK-PEG and PLGA-TK-PEG-PBO nanoparticles in the embodiments of the present invention. Figure 2 A represents the particle size distribution of blank PLGA-TK-PEG nanoparticles. Figure 2 B represents the particle size distribution of PLGA-TK-PEG nanoparticles loaded with PBO (i.e., PLGA-TK-PEG-PBO). Figure 2 C represents the Zeta potential diagram. Figure 2 D represents the Fourier transform infrared spectrum.

[0032] Figure 3 The figures show the in vitro cumulative release curves of PBO in PLGA-TK-PEG-PBO under different ROS conditions in this invention embodiment. The data shown in the figures are the mean ± standard deviation.

[0033] Figure 4 The figure shows the inhibition time of P450 enzyme by free PBO and PLGA-TK-PEG-PBO as synergists for high-efficiency cypermethrin in this embodiment of the invention. In the figure, different lowercase letters between different treatments at the same detection time represent the results of significance analysis. P <0.05.

[0034] Figure 5This figure compares the toxicity enhancement of free PBO and PLGA-TK-PEG-PBO as synergists for high-efficiency cypermethrin against aphids in this invention embodiment. In the figure, different lowercase letters between different treatments at the same detection time represent the results of significance analysis. P <0.05.

[0035] Figure 6 The figure shows the change in the relative intensity of reactive oxygen species after treatment with high-efficiency cypermethrin in this embodiment of the invention. In the figure, ** indicates the significance analysis results for the same detection time. P <0.01. Detailed Implementation

[0036] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0037] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0038] Piperyl butyl ether (PBO, CAS No. 51-03-6) in the following examples is a product of Shanghai Yuanye Biotechnology Co., Ltd., catalog number B67398.

[0039] The carrier PLGA-TK-PEG in the following examples is a triblock copolymer and is a product of Xi'an Ruixi Biotechnology Co., Ltd.

[0040] The dichloromethane (CAS No.: 75-09-2) used in the following examples is a product of Sangon Biotech (Shanghai) Co., Ltd., with catalog number A530003.

[0041] The PVA (polyvinyl alcohol, CAS No.: 9002-89-5) used in the following examples is a product of Sangon Biotech (Shanghai) Co., Ltd., with item number A420633.

[0042] The cypermethrin-resistant *Aphidius gracilis* in the following examples were obtained by continuously screening *Aphidius gracilis* with cypermethrin for more than 3 years under indoor conditions (24 ± 1 ℃, 60 ± 10% RH, 16:8 h L:D), as described in the non-patent literature "Yan Juncheng, P450 Mediating the resistance mechanism of *Aphidius gracilis* to cypermethrin, 2024. China Agricultural University, Master's Thesis". The public can obtain the paper from the applicant to replicate the experiments of this application.

[0043] The high-efficiency cypermethrin used in the following examples is a product of Jiangsu Changlong Agrochemical Co., Ltd.

[0044] The approval number of the dwarf 58 in the following examples is National Approval Wheat 2005008, and it is commercially available.

[0045] Example 1: Preparation and characteristics of reactive oxygen species-responsive nano-piperidine butyl ether synergist 1. Composition The reactive oxygen species responsive nano-piperyl butyl ether synergist of the present invention is named PLGA-TK-PEG-PBO, which is composed of nano-carrier PLGA-TK-PEG and its encapsulated piperin butyl ether.

[0046] The specific components are as follows: The carrier, PLGA-TK-PEG, is a core-shell nanoparticle, a triblock copolymer composed of a hydrophilic PEG (polyethylene glycol) shell, a hydrophobic PLGA (polylactic acid-glycolic acid copolymer) core, and a ROS (reactive oxygen species) responsive switch TK (thioketal bond). During the evaporation of organic solvents, PLGA-TK-PEG spontaneously assembles into core-shell nanoparticles. The hydrophobic PLGA-TK segments aggregate inward to form a dense core, serving as a reservoir for the hydrophobic piperonyl butyl ether; while the hydrophilic PEG segments extend outward, forming a hydrated steric hindrance layer in the aqueous environment, thus constituting the core and shell.

[0047] Active ingredient: Piperyl butyl ether (PBO, CAS No. 51-03-6), encapsulated inside and on the surface of the carrier.

[0048] 2. Preparation method (emulsion-solvent evaporation method) 1) Weigh 50 mg of PLGA-TK-PEG and 2.65 mg of PBO, and dissolve them in 1 mL of dichloromethane; 2) Add 5 mL of 5% PVA (polyvinyl alcohol) aqueous solution and sonicate with a 300 W probe for 10 min. 3) Stir at 500 rpm for 1 h at room temperature to evaporate the organic solvent; 4) Centrifuge at 4000 rpm for 5 min to remove large particles and collect the supernatant; 5) Centrifuge the supernatant at 13000 rpm for 5-10 min and collect the precipitate; 6) After precipitating and washing twice with water, add 5% trehalose (a product of Sangon Biotech (Shanghai) Co., Ltd., catalog number A631024) as a freeze-drying protectant, and freeze-dry (pre-freeze at -80℃ for 4 h, then place in a freeze dryer for primary drying at -40℃ for 24 h, and then secondary drying at 25℃ for 6-8 h). The resulting white powder is nanoparticle, which is the reactive oxygen species responsive nano-piperidine butyl ether synergist PLGA-TK-PEG-PBO of this invention.

[0049] 3. Particle size, PDI, and zeta potential measurement Transmission electron microscopy (TEM) images of PLGA-TK-PEG-PBO and the original PLGA-TK-PEG are shown below. Figure 1 .

[0050] The results showed that the original PLGA-TK-PEG nanoparticles ( Figure 1 A) and PLGA-TK-PEG nanoparticles loaded with PBO ( Figure 1 Both samples (B) exhibited a uniform spherical morphology with a smooth surface. Notably, no significant morphological differences or aggregation were observed between the blank carrier and the drug-loaded nanoparticles, indicating that the encapsulation of hydrophobic PBO did not disrupt the structural integrity of the polymer matrix or the self-assembly process. Both samples displayed clear, discrete spherical structures, confirming that both formulations successfully formed stable nanoparticles.

[0051] The particle size distributions of blank PLGA-TK-PEG nanoparticles and PBO-loaded PLGA-TK-PEG nanoparticles (i.e., PLGA-TK-PEG-PBO) were determined using a nanoparticle size analyzer (NanoBrook 90plus PALS, Brookhaven, USA). (See attached figures). Figure 2 A and Figure 2 The B. Zeta potential diagram is shown below. Figure 2 C.

[0052] The results showed that the average particle size of the blank PLGA-TK-PEG nanoparticles was 140.127±5.15 nm; the PDI (Polymer Dispersity Index) was 0.197±0.01; and the Zeta potential was -13.483±3.00 mV. The average particle size of the PBO-loaded PLGA-TK-PEG nanoparticles (i.e., PLGA-TK-PEG-PBO) was 136.31±7.01 nm; the PDI was 0.159±0.05; and the Zeta potential was 2.604±0.10 mV.

[0053] Fourier transform infrared spectra were measured using an IRspirit-X spectrometer (Shimadzu, Japan). The results are shown below. Figure 2 The successful encapsulation of PBO in PLGA-TK-PEG nanoparticles was verified by Fourier transform infrared spectroscopy comparison analysis. The spectral characteristics of the PLGA+PEG physical mixture showed a characteristic peak at 1700 cm⁻¹. - ¹(ester bond C=O), 1455 cm - ¹(CH2 bending) and 1385 cm - ¹(CH3 deformation). PLGA-TK-PEG support spectra in the range of 500–750 cm⁻¹ - ¹A new peak appeared in the fingerprint region, confirming the successful introduction of the thioketal (TK) bond. The spectrum of the drug-loaded nanoparticles differed significantly from that of the blank carrier, and the newly added characteristic peak corresponded perfectly to the PBO structure: 1490 cm⁻¹ - ¹(Benzene ring C=C), 1250 cm - ¹(aryl ether COC) and 925 cm - ¹(methylenedioxyO-CH2-O deformation) indicates that PBO has been chemically embedded in the polymer network, rather than through surface adsorption or physical mixing, providing conclusive chemical evidence for successful drug encapsulation.

[0054] The above test results all indicate that the prepared PLGA-TK-PEG-PBO nanoparticles have uniform particle size, good dispersibility, and high stability.

[0055] 4. Determination of drug loading and encapsulation efficiency When determining the drug loading and encapsulation efficiency of nanoparticles using HPLC (C18 column, mobile phase: acetonitrile and water), the free drug is first separated by ultrafiltration and centrifugation. The total drug content (W1) is then extracted by ultrasonication after dissolving and destroying the nanoparticles, and the content is calculated using a standard curve with linearity R² ≥ 0.999. The encapsulation efficiency EE and drug loading DL are calculated according to formulas (1) and (2), respectively: Encapsulation efficiency EE(%) = (W1- W2) / W0× 100% (1).

[0056] Drug loading DL(%) = (W1- W2) / M × 100% (2).

[0057] In the above formula, W0 represents the total mass of the drug added, which is the total mass of drug PBO initially added to the system during the preparation of PLGA-TK-PEG-PBO. W1 represents the total mass of all drug PBO in the system, including drug PBO encapsulated in nanoparticles and free drug PBO in solution; W2 represents the mass of free drug PBO separated by ultrafiltration and centrifugation that did not enter the nanoparticles; and M represents the mass of the nanocarrier PLGA-TK-PEG.

[0058] The results showed that the prepared PLGA-TK-PEG-PBO had a drug loading of 10.6% and an encapsulation rate of 88.5%, meeting the requirements for efficient delivery and field application.

[0059] 5. Measurement of ROS response and release behavior The above PLGA-TK-PEG-PBO was added to 0.01 M PBS (phosphate buffer) at pH 7.4, setting up two environments: Normal environment: No H2O2.

[0060] ROS environment: Add H2O2 to the PBS containing PLGA-TK-PEG-PBO to achieve a concentration of 1 mM (millimoles per liter).

[0061] The PBO release was detected, and the results are shown below. Figure 3 In a normal environment, PBO release is slow; in a ROS environment, the PBO release rate increases significantly within 24 hours, confirming that the nanoparticles have typical ROS-responsive release characteristics.

[0062] The ROS-responsive release mechanism of PLGA-TK-PEG-PBO in this invention is as follows: Under normal conditions, TK is stable, the PLGA-TK-PEG-PBO nanoparticles remain intact, and PBO is released at a low rate. In a ROS environment (such as when the H2O2 concentration reaches 1 mM), the TK bond breaks, the carrier degrades, and the PBO in PLGA-TK-PEG-PBO is rapidly released within 24 h, achieving intelligent on-demand release.

[0063] 6. Application of resistance control The PLGA-TK-PEG-PBO of this application can be compounded with high-efficiency cypermethrin for the control of cypermethrin-resistant aphids, especially suitable for field populations with a resistance multiple ≥ 1000 times. Specific efficacy verification is shown in Example 2.

[0064] Based on the above results, PLGA-TK-PEG-PBO was selected as the product, and the relevant information is as follows: Product composition: PLGA-TK-PEG was used as a carrier, with TK (thioacetal bond) on the carrier serving as the ROS-responsive site; Piperyl butyl ether is used as an active synergist, which is encapsulated inside and on the surface of the carrier.

[0065] Key performance: Particle size: 130–150 nm; PDI: ≤0.2; Zeta potential: 25–30 mV; Drug loading: >10%; Encapsulation rate: ≥85%; Core Mechanism: Under normal conditions: Nanoparticles are stable, PBO is released slowly, reducing loss and degradation; ROS enrichment environment (pesticide stress): The thioketal bonds of the carrier break, the nanoparticles disintegrate, and PBO is released rapidly and in large quantities, continuously inhibiting P450 detoxification enzymes and achieving precise synergistic effects.

[0066] Example 2: Synergistic effect of reactive oxygen species-responsive nano-piperidine butyl ether synergist against resistant aphids The test insect source was the wingless adult *Aphidius gracilis* resistant to cypermethrin. Under indoor conditions (24 ± 1 ℃, 60 ± 10% RH, 16:8 h L:D), the insects were continuously screened with cypermethrin for more than 3 years (see non-patent literature "Yan Juncheng, P450-mediated resistance mechanism of *Aphidius gracilis* to cypermethrin, 2024. China Agricultural University, Master's Thesis").

[0067] The toxicity of lambda-cypermethrin to sensitive (S) and resistant (R) strains was determined using a leaf-immersion assay with aphids present. Freshly cut 3 cm diameter wheat leaf segments were placed in petri dishes lined with moistened filter paper, and 20 wingless adult aphids of similar size were inoculated into each segment. Lambda-cypermethrin was first dissolved in acetone to prepare a high-concentration stock solution, which was then diluted with distilled water containing 0.1% Triton X-100 to prepare five concentration gradients of lambda-cypermethrin solutions (5000 mg / L, 8000 mg / L, 10000 mg / L, 20000 mg / L, and 40000 mg / L). The leaf segments with aphids were immersed in the test solution for 5 seconds, and excess solution was gently blotted out with filter paper before being returned to the corresponding petri dish. Each concentration was replicated three times, with distilled water containing 0.1% Triton X-100 serving as a control. Mortality was assessed 48 hours after application. The bioassay data were analyzed using POLO Plus (LeOra Software) to calculate the median lethal concentration (LC50). 50 ) and its 95% confidence interval (CI), toxicity regression line slope, and chi-square value. The resistance ratio (RR) is calculated according to formula (3): RR = LC 50 (Resistant strain) / LC 50 (Sensitive strains) (3).

[0068] The results showed that the LC50 of the highly efficient cypermethrin-resistant strain (R) was... 50 =11320.07 mg / L, resistance multiple 1144.4 times.

[0069] The above results confirm that the lambda-eating aphid resistant to high-efficiency cypermethrin used in this embodiment is resistant to high-efficiency cypermethrin and can be used for subsequent efficacy verification of synergists.

[0070] 1. Significantly reduces the toxicity of resistant aphids to highly effective cypermethrin. Four groups were established to screen wingless adult *Aphidius gracilis* resistant for more than 3 years under indoor conditions (24 ± 1 ℃, 60 ± 10% RH, 16:8 h L:D): 1-1. High-efficiency cypermethrin (single agent); 1-2. High-efficiency cypermethrin + PBO (free PBO control); 1-3, High-efficiency cypermethrin + PLGA-TK-PEG (blank vector control); 1-4. High-efficiency cypermethrin + PLGA-TK-PEG-PBO (the reactive oxygen species responsive nano-piperidine butyl ether synergist of the present invention, prepared from Example 1); In the free PBO group, PBO stock solution dissolved in acetonitrile was diluted with distilled water containing 0.1% Triton X-100 to a final concentration of 250 mg / L (organic phase content ≤1%). In the blank PLGA-TK-PEG nanoparticle and drug-loaded PLGA-TK-PEG-PBO nanoparticle groups, 2.5 mg of lyophilized blank polymer powder was resuspended in 10 mL of distilled water containing 0.1% Triton X-100, and after sonication in an ice bath, a colloidal dispersion with a final concentration of 250 mg particles / L was obtained.

[0071] The toxicity of four pesticide combinations to a resistant strain of *Aphidius gracilis* (denoted as R) was determined using the leaf immersion assay with insects present. Each pesticide combination was configured with five gradients based on the final concentration of high-efficiency cypermethrin. Specifically: 1-1, the high-efficiency cypermethrin (single agent) group had five concentrations of 5000 mg / L, 8000 mg / L, 10000 mg / L, 20000 mg / L, and 40000 mg / L; 1-2, the high-efficiency cypermethrin + PBO (250 mg / L) group had five concentrations of 500 mg / L, 1000 mg / L, 2000 mg / L, 4000 mg / L, and 8000 mg / L; 1-3, the high-efficiency cypermethrin + PLGA-TK-PEG (250 mg / L) group had five concentrations of 2000 mg / L, 4000 mg / L, 8000 mg / L, 16000 mg / L, and 32000 mg / L; 1-4, the high-efficiency cypermethrin + PLGA-TK-PEG-PBO (250 mg / L) group had five concentrations of 2000 mg / L, 4000 mg / L, 8000 mg / L, 16000 mg / L, and 32000 mg / L; The five concentrations (50 mg / L, 100 mg / L, 200 mg / L, 400 mg / L, and 800 mg / L) were set in the mg / L group; their LC50 was determined. 50 To evaluate the synergistic effect of PBO delivery via nanocarrier on high-efficiency cypermethrin.

[0072] Each treatment had 3 replicates, with 15 aphids per replicate per group.

[0073] Table 1: Comparison of the synergistic effects of PBO and PLGA-TK-PEG-PBO on high-efficiency cypermethrin

[0074] In Table 1, * represents the results of the significance analysis. P <0.05.

[0075] The results are shown in Table 1: In treatments 1-4, PLGA-TK-PEG-PBO, when combined with 50 mg / L, 100 mg / L, 200 mg / L, 400 mg / L, and 800 mg / L of lambda-cypermethrin, all enhanced the control effect against resistant strains of *Cephalotaxus fortunei*. The LC50 of lambda-cypermethrin was measured using PLGA-TK-PEG-PBO. 50 The concentration was reduced to 223.7 mg / L, with a synergistic effect ratio (SR) of 54.22, which was much higher than that of free PBO in groups 1-2 (SR=7.44) and blank vector in groups 1-3 (SR=1.23).

[0076] Therefore, when used in combination with PLGA-TK-PEG-PBO, the working concentration of the high-efficiency cypermethrin can be 50 mg / L - 800 mg / L. Considering maximizing the control effect while minimizing pesticide residues, it is recommended that the working concentration of the high-efficiency cypermethrin when used in combination with PLGA-TK-PEG-PBO be 200 mg / L - 400 mg / L, with the optimal working concentration being 200 mg / L - 400 mg / L.

[0077] 2. Synergistic effect when used in combination with high-efficiency cypermethrin (223.7 mg / L) This study investigated the synergistic effect of the combined use of PLGA-TK-PEG-PBO of this invention with high-efficiency cypermethrin (223.7 mg / L).

[0078] To investigate wingless adult *Aphidius gracilis* resistant to continuous screening with high-efficiency cypermethrin for more than 3 years under indoor conditions (24 ± 1 ℃, 60 ± 10% RH, 16:8 h L:D), the mortality rate of the resistant strain (denoted as R) was determined using the leaf-dip assay with insects at a concentration of 223.7 mg / L of high-efficiency cypermethrin, while simultaneously using the following four reagents in combination: 2-1, 0.1% Triton X-100 distilled water (control); 2-2, PLGA-TK-PEG (blank vector control); 2-3, PBO (free PBO control); 2-4, PLGA-TK-PEG-PBO; The final concentrations of PBO, PLGA-TK-PEG, and PLGA-TK-PEG-PBO were all 250 mg / L.

[0079] Each treatment had 3 replicates, with 15 aphids per replicate per group.

[0080] The aphid mortality rate was measured at 3 h, 6 h, 12 h, 24 h, and 48 h after application of the pesticide. The results are shown in [the table below]. Figure 5 : Three hours after application: As a synergist for high-efficiency cypermethrin, PLGA-TK-PEG-PBO (groups 2-4) achieved a 20% aphid mortality rate, significantly higher than that of the free PBO group (groups 2-3, 3.33%), the blank carrier group (groups 2-2, 2.5%), and the 0.1% Triton X-100 distilled water control group (group 2-1, 0.83%). This indicates that PLGA-TK-PEG-PBO of the present invention significantly overcomes the technical bottleneck of slow onset and insufficient initial synergistic effect of free PBO, and achieves rapid knockdown and immediate control of aphids when used in combination with low concentrations of high-efficiency cypermethrin.

[0081] Six hours after application: As an synergist for high-efficiency cypermethrin, the aphid mortality rate in the PLGA-TK-PEG-PBO group (groups 2-4) increased to about 37.5%, which was much higher than that in the free PBO group (groups 2-3, 5%), the blank carrier group (groups 2-2, 4.17%), and the 0.1% Triton X-100 distilled water control group (groups 2-1, 4.17%). 12 hours after application: As an synergist for high-efficiency cypermethrin, the aphid mortality rate in the PLGA-TK-PEG-PBO group (groups 2-4) increased to about 47.5%, which was much higher than that in the free PBO group (groups 2-3, 12.5%), the blank carrier group (group 2, 5%), and the 0.1% Triton X-100 distilled water control group (group 2-1, 5.83%). 24 hours after application: As a synergist for high-efficiency cypermethrin, the aphid mortality rate in the PLGA-TK-PEG-PBO group (groups 2-4) reached 70%, while that in the free PBO group (groups 2-3) was only 14.17%, with a synergistic effect of more than 5 times. 48 hours after application: As a synergist for high-efficiency cypermethrin, the aphid mortality rate in the PLGA-TK-PEG-PBO group (groups 2-4) remained above 70%, while that in the free PBO group (groups 2-3) was only 16.67%. This indicates that the synergist of the present invention achieves long-lasting and continuous synergistic effects, breaking through the technical bottleneck of the narrow synergistic window of free PBO.

[0082] Example 3: Determination of the time-dependent inhibition of P450 enzyme The test material was wingless adult aphids of the grain-resistant aphid (same as Example 2) that had been continuously screened for more than 3 years with high efficiency cypermethrin under indoor conditions (24 ± 1 ℃, 60 ± 10% RH, 16:8 h L:D).

[0083] Resistant aphids were treated with distilled water containing 0.1% Triton X-100, free PBO, PLGA-TK-PEG (blank carrier control), and PLGA-TK-PEG-PBO (i.e., the reactive oxygen species-responsive nano-piperidine butyl ether synergist of this invention). For the free PBO group, a PBO stock solution dissolved in acetonitrile was diluted to a final concentration of 250 mg / L (organic phase ≤1%) with distilled water containing 0.1% Triton X-100. For the blank PLGA-TK-PEG nanoparticles and the drug-loaded PLGA-TK-PEG-PBO nanoparticles, 2.5 mg of lyophilized blank polymer powder was resuspended in 10 mL of distilled water containing 0.1% Triton X-100, and after sonication in an ice bath, a colloidal dispersion with a final concentration of 250 mg particles / L was obtained. Resistant aphids were treated using the leaf-immersion method with insects, with three replicates for each treatment and 15 aphids per replicate per group.

[0084] The changes in P450 enzyme activity of resistant aphids in each group were measured at 3 h, 6 h, 12 h, 24 h, and 48 h after treatment (method referenced "Yan Juncheng, P450-mediated resistance mechanism of *Aphidius gracilis* to lambda-cyhalothrin, 2024. Master's thesis, China Agricultural University." 7-ethoxycoumarin used was purchased from Sigma-Aldrich, USA). Results are shown below. Figure 4 : Free PBO: The enzyme activity was basically restored after 24 h, with an activity of 31.29 pmol / min•mg protein.

[0085] PLGA-TK-PEG-PBO: It still maintained extremely strong inhibition at 24 h and 48 h, with enzyme activities of 13.41 and 18.56 pmol / min•mg protein, respectively.

[0086] This demonstrates that the PLGA-TK-PEG-PBO of the present invention can effectively inhibit detoxification enzymes for a long time, overcoming the short-term limitation of traditional PBO being easily metabolized in pests.

[0087] Example 4: Verification of ROS Levels and Release Timing To verify whether the mechanism of action of the nano-synergist is temporally related to the accumulation of reactive oxygen species (ROS), this study examined the changes in ROS levels in *Triticum aphidii* (treated with 223.7 mg / L of pesticide using the leaf-dip method with insects present) after treatment with lambda-cypermethrin. A control group treated with 0.1% Triton X-100 distilled water was included as a blank control (CK). Ten biological replicates were set up for each group.

[0088] ROS detection procedure: The ROS assay was performed using a commercially available ROS detection kit (Shanghai Bebo Biotechnology Co., Ltd., product number: BB-470515). The specific procedures are as follows: 1) Sample preparation: Take 50 mg of wingless adult aphids and rinse them with PBS buffer.

[0089] 2) Homogenization and centrifugation: Place the sample in a glass homogenizer, add 500 μL of buffer solution for homogenization; then centrifuge at 4℃ and carefully aspirate the supernatant.

[0090] 3) Probe incubation: Take 200 μL of the above supernatant, add 2 μL of specific fluorescent probe, and incubate in a dark environment at 37℃ for 30 minutes.

[0091] 4) Fluorescence detection: Fluorescence intensity was measured using a BioTek FLx800 multi-functional microplate reader (USA).

[0092] 5) Data Processing: ROS intensity was normalized using protein concentration based on the average fluorescence intensity of the control group (CK) at corresponding time points to eliminate errors caused by sample size differences. Data are expressed as mean ± standard error (Mean ± SEM). Significance between groups was analyzed using a t-test, with ** indicating statistical significance. P <0.01.

[0093] The results are as follows Figure 6 As shown, compared with the control group, the ROS level in wheat aphids showed a significant and continuous increasing trend after treatment with high-efficiency cypermethrin. At 3 h, 6 h, and 12 h, the relative ROS intensity of the treatment group was 2.3, 2.3, and 2.4 times that of the control group, respectively; until 24 h and 48 h, although the ROS level of the treatment group decreased, it was still 2.0 and 1.7 times that of the control group.

[0094] The above results indicate that treatment with highly efficient cypermethrin can continuously induce high levels of oxidative stress in wheat aphids for up to 48 hours. Combined with previous experimental results, the peak release of nano-PBO highly coincides with this period of ROS elevation. This perfect temporal match demonstrates that the nanocarrier can precisely respond to the insecticide-induced ROS burst signal, achieving "on-demand release" of the drug, thereby maximizing the synergistic effect of the synergist and insecticide in the temporal dimension (targeting-time-concentration triple synergy), ultimately effectively overcoming aphid resistance.

[0095] Example 5: Synergistic effect of reactive oxygen species-responsive nano-piperidine butyl ether synergist in the control of wheat aphids in the field. This embodiment is used to verify the synergistic effect of the reactive oxygen species-responsive nano-piperidine butyl ether synergist (PLGA-TK-PEG-PBO) on the control of wheat aphids by high-efficiency cypermethrin in actual field applications.

[0096] The field trial was conducted in Zhuozhou City, Hebei Province, China in April 2024, with winter wheat as the test crop. Triticum aestivum cv. dwarf resistant 58). The experimental plots were kept in a natural occurrence state, with the dominant species being the cereal tube aphid (Cephalotaxus fortunei). Rhopalosiphum padi ) and wheat aphid ( Sitobion avenae To ensure the reliability of the efficacy evaluation, no insecticides or systemic pesticides were applied for at least 15 days prior to the trial. A pre-application survey confirmed that the aphid population in the field exceeded 500 aphids per 100 plants, meeting the requirements for wheat aphid control trials as stipulated in the Chinese National Standard "Guidelines for Field Efficacy Tests of Pesticides" (GB / T 17980.79-2004).

[0097] The experiment consisted of four treatment groups and one blank control group, as detailed below: Treatment Group 1 (Nano-synergist Group): High-efficiency cypermethrin + reactive oxygen species responsive nano-synergist of this invention (i.e., high-efficiency cypermethrin + PLGA-TK-PEG-PBO); Treatment group 2 (blank nanocarrier group): high-efficiency cypermethrin + blank carrier (i.e., high-efficiency cypermethrin + PLGA-TK-PEG); Treatment group 3 (free PBO control group): high-efficiency cypermethrin + free piperonyl butyl ether (i.e., high-efficiency cypermethrin + PBO); Treatment group 4 (single-agent group): only high-efficiency cypermethrin; Blank control group: Water (CK).

[0098] Each treatment was replicated five times, with plots measuring 3 m × 2 m, using a randomized block design. A PBO to lambda-cyhalothrin (PBO) ratio of 1.12:1 was used for field spraying. During application, the pesticides for each treatment were mixed thoroughly in a backpack sprayer. 20 g of PBO active ingredient and 22.4 g of PBO (or an equivalent nano-formulation) active ingredient were applied per hectare.

[0099] The number of surviving aphids was investigated before application and 3 and 7 days after application. Ten wheat plants were randomly selected from each plot, and the number of live aphids per plant was recorded using the direct counting method.

[0100] The prevention and control effect is calculated using formulas (4) and (5): Population decline rate (%) = [(Number of insects before application - Number of insects after application) / Number of insects before application] × 100 (4).

[0101] Prevention and control effect (%) = [(reduction rate of treatment group - reduction rate of control group) / (100 - reduction rate of control group)] × 100 (5).

[0102] The results of the field trials are shown in Table 2, which verify the practical application effect of the nano-synergist of the present invention in the field: Table 2. Field control effects of different treatments on wheat aphids

[0103] Note: Data in Table 2 are expressed as mean ± standard error (Mean ± SEM). Significance of differences between groups was analyzed using t-tests, with ** indicating statistical significance. P <0.01, ns indicates no significant difference.

[0104] The effect of single-agent control was limited: the population decline rate of the commercial standard agent 4.5% high-efficiency cypermethrin EC 3 days and 7 days after application was 40.58% and 49.21%, respectively, and the corresponding corrected control effect was only 51.19% and 60.30%.

[0105] The blank carrier showed no significant synergistic effect: when high-efficiency cypermethrin was used in combination with the blank PLGA carrier, only a slight synergistic effect was observed, with the control effect ranging from 54.20% to 59.64%, indicating that the carrier material itself did not have significant insecticidal activity.

[0106] Free PBO has a certain synergistic effect: when free PBO is used in combination with high-efficiency cypermethrin, the control effect is increased to 64.45% and 61.38% 3 days and 7 days after application, respectively, which confirms the effectiveness of PBO as a synergist in the field.

[0107] The nano-synergist of this invention exhibits significant effects: At the same dosage, the application of the reactive oxygen species-responsive nano-piperidine butyl ether synergist (PLGA-TK-PEG-PBO) described in this invention demonstrates the superior field control efficacy. Three days after application, the population reduction rate reached 64.55%, with a control efficacy of 70.87%; by seven days after application, the population reduction rate further increased to 79.41%, with a control efficacy as high as 84.06%.

[0108] The above results demonstrate that the reactive oxygen species-responsive nano-piperyl butyl ether synergist PLGA-TK-PEG-PBO of this invention has the following advantages over free piperin butyl ether: enhanced rapid action, significantly improved stability, intelligent ROS-responsive release, ultra-high resistance reversal ability, and a synergistic effect with highly effective cypermethrin reaching 54.22, effectively overcoming the ultra-high level of resistance (>1000 times) of aphids to highly effective cypermethrin, achieving highly efficient control of resistant populations. Furthermore, it can be delivered in a targeted manner, the carrier is biodegradable, and PBO is released on demand, reducing dosage and environmental residue.

[0109] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A complex, characterized in that, The complex is a reactive oxygen species-responsive nano-piperyl butyl ether synergist, composed of PLGA-TK-PEG and its encapsulated piperin butyl ether, with a drug loading of 8%-12%.

2. The complex according to claim 1, characterized in that, The hydrophobic segment PLGA-TK of the PLGA-TK-PEG aggregates inward to encapsulate the hydrophobic piperin butyl ether, while the hydrophilic segment PEG forms a core-shell structure on the outside.

3. A method for preparing the complex according to claim 1 or 2, characterized in that, The method includes the steps of adding the PLGA-TK-PEG and the piperonyl butyl ether to an organic solvent, adding a surfactant, emulsifying, then evaporating the organic solvent and collecting the precipitate to obtain the complex; The organic solvent is at least one of dichloromethane and trichloromethane; the surfactant is a nonionic surfactant selected from at least one of polyvinyl alcohol, polyoxyethylene sorbitan monooleate, and polyoxyethylene polyoxypropylene block copolymer.

4. The method according to claim 3, characterized in that, The organic solvent is dichloromethane, and the surfactant is polyvinyl alcohol; the method further includes the step of freeze-drying the precipitate.

5. The application of the complex according to claim 1 or 2 in enhancing the aphid control effect of cypermethrin.

6. The application according to claim 5, characterized in that, The aphids mentioned are the cereal constrictor aphid and / or the wheat long-tubed aphid.

7. The application according to claim 5 or 6, characterized in that, The aphids mentioned are those resistant to highly effective cypermethrin.

8. The application according to claim 7, characterized in that, The complex: the mass ratio of the high-efficiency cypermethrin used is 1.12:1; the working concentration of the high-efficiency cypermethrin is 50 mg / L - 800 mg / L.

9. The application according to claim 8, characterized in that, The working concentration of the high-efficiency cypermethrin is 200 mg / L-400 mg / L.

10. The application according to claim 9, characterized in that, The working concentration of the high-efficiency cypermethrin is 223.7 mg / L.

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