Insecticide delivery preparation based on binary synergistic nanoparticles and preparation method thereof

Abstract

The invention provides an insecticide delivery preparation based on binary synergistic nanoparticles and a preparation method thereof, an IMI (at) AgNPs (at) CMSNs compound is prepared, and the binary system remarkably overcomes the drug resistance of cotton aphid to imidacloprid by integrating silver nanoparticles (AgNPs) and mesoporous silica nanoparticles (MSNs).

Description

Technical Field

[0001] This invention relates to the field of insecticide technology, specifically to an insecticide delivery formulation based on binary synergistic nanoparticles and its preparation method. Background Technology

[0002] Cotton aphid (scientific name: ( Aphis gossypii The cotton aphid (Glover), belonging to the family Aphididae in the order Hemiptera, is a pest that feeds on cotton tree sap and severely impacts global cotton production. It causes significant economic losses to the cotton industry through direct feeding, virus transmission, and contamination of honeydew secreted by aphids. Currently, the cotton aphid is a major pest in cotton cultivation, and in China, controlling this aphid still relies heavily on the irreplaceable traditional method of insecticide application. Neonicotinic insecticides are widely used in pest control due to their effectiveness against sucking pests and low toxicity to mammals. However, due to increasing pressure from resistance selection, cotton aphids have gradually developed resistance to many neonicotinic insecticides. Summary of the Invention

[0003] In view of the problems pointed out in the background art, the present invention proposes an insecticide delivery formulation based on binary synergistic nanoparticles and its preparation method to solve the above-mentioned technical problems.

[0004] The technical solution of this invention is implemented as follows: An insecticide delivery formulation based on binary synergistic nanoparticles combines silver nanoparticles (AgNPs) and carboxyl-functionalized mesoporous silica nanoparticles (CMSNs) to obtain the complex AgNPs@CMSNs.

[0005] The present invention is further configured to combine the complex AgNPs@CMSNs with imidacloprid to obtain the complex IMI@AgNPs@CMSNs.

[0006] A method for preparing an insecticide delivery formulation based on binary synergistic nanoparticles includes the following steps: Step 1: Dissolve 0.5 mg CMSNs in 5 mL of phosphate-buffered saline (PBS) at pH 7.4, and add 0.5 mg AgNPs; Step 2: Sonicate the mixture for 10 min and then vibrate it at 200 rpm for 4 h; Step 3: Recover AgNPs@CMSNs nanoparticles by centrifugation at 10,000 rpm for 5 min; Step 4: Wash with deionized water and dry under vacuum to obtain the complex AgNPs@CMSNs.

[0007] The present invention is further configured to include the following steps: Step 5: Dissolve imidacloprid in acetone to obtain a stock solution (10,000 mg / L), then adjust to 5,000 mg / L with distilled water; Step 6: Suspend 1 mg AgNPs@CMSNs in 10 mL of 5000 mg / L imidacloprid solution, sonicate the mixture for 10 min and shake at 200 rpm for 4 h to obtain the complex IMI@AgNPs@CMSNs.

[0008] By adopting the above technical solution, the beneficial effects of the present invention are as follows: The present invention provides an insecticide delivery formulation based on binary synergistic nanoparticles and its preparation method, which prepares an IMI@AgNPs@CMSNs complex. This binary system significantly improves the effectiveness of insecticides against the insecticide-resistant pest, the sweet cotton aphid, by integrating silver nanoparticles (AgNPs) and mesoporous silica nanoparticles (MSNs). Attached Figure Description

[0009] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0010] Figure 1 : A schematic diagram of the synthesis of the AgNPs@CMSNs-mediated imidacloprid delivery system.

[0011] (A, C) Scanning electron microscope images of AgNPs@CMSNs and IMI@AgNPs@CMSNs.

[0012] (B, D) Dark-field scanning electron microscopy images and energy dispersive spectroscopy characterization of AgNPs@CMSNs and IMI@AgNPs@CMSNs.

[0013] (E) Particle size distribution of AgNPs, CMSNs, AgNPs@CMSNs and IMI@AgNPs@CMSNs.

[0014] (F) Zeta potential data for AgNPs, CMSNs, AgNPs@CMSNs and IMI@AgNPs@CMSNs.

[0015] Fourier transform infrared spectra of (G)CMSNs(I), imidacloprid(II) and IMI@AgNPs@CMSNs(III).

[0016] (H) Adsorption capacity of AgNPs@CMSNs and IMI@AgNPs@CMSNs for imidacloprid. The bar charts labeled with different lowercase letters represent the significant differences shown by Tukey's multiple comparison test after one-way ANOVA. * P <0.05).

[0017] Figure 2 : The effects of AgNPs@CMSNs treatment on the gut symbiotic flora of four field populations of cotton aphid.

[0018] (A) It belongs to the top ten gut bacteria in terms of relative abundance.

[0019] (B) The heatmap shows the changes in the abundance of the top ten gut symbiotic genera after treatment with AgNPs@CMSNs.

[0020] (C) Principal component analysis showed differences in the gut microbiota composition of cotton aphids.

[0021] (DG) Differences in the abundance of gut bacteria genera between the AgNPs@CMSNs treatment group and the control group based on the T-test.

[0022] (HI) The diversity and richness of gut symbiotic bacteria were analyzed by Shannon index and Chao 1 index respectively (t test).

[0023] (JK) Relative abundance of Oligotrophomonas and Sphingomonas in the intestines of cotton aphids (t-test, * P<0.05).

[0024] Figure 3 : The effects of AgNPs@CMSNs' antibacterial activity against gut symbiotic bacteria and its metabolic capacity on imidacloprid.

[0025] (A, B) bar charts show the diameters of the inhibition zones of AgNPs and AgNPs@CMSNs against Oligotrophozoites and Sphingosine Monoclonal antibodies, respectively (Student t-test). * P <0.05).

[0026] (C, D) Minimum inhibitory concentrations (MICs) of AgNPs@CMSNs against Oligotrophomonas and Sphingosine Monoclonal.

[0027] (E) Changes in the concentration of imidacloprid and its metabolites during 6 days of co-culture with oligotrophomonas.

[0028] (F) Phylogenetic relationships of symbiotic oligotrophomonas strains. Blue asterisks indicate oligotrophomonas strains isolated from the intestines of cotton aphids (Aphisgossypii).

[0029] (G) Inhibition zone diameters of AgNPs (a) and AgNPs@CMSNs (b) on agar plates against oligotrophomonas and sphingosine monoclonal antibodies.

[0030] (HK) Metabolites produced by the degradation of imidacloprid by oligotrophic monocytes, obtained by high performance liquid chromatography-mass spectrometry (HPLC-MS).

[0031] (L) A hypothetical pathway for the degradation of imidacloprid by oligotrophomonas.

[0032] Figure 4 : Study on wetting characteristics and adhesion properties of IMI@AgNPs@CMSNs nanocomposites.

[0033] (A) Schematic diagram of the experimental design of IMI@AgNPs@CMSNs nanocomposite materials.

[0034] (B, C) Scanning electron microscopy images of different samples on the surface of cotton leaves before and after washing.

[0035] (D) Recovery rate data for IMI.

[0036] (E) Contact angle measurements of different solutions on cotton leaf surfaces. Data from each group were analyzed using one-way ANOVA and Tukey's multiple comparison test (significance level). * P A value <0.05 indicates a significant difference.

[0037] Figure 5 : The permeability and insecticidal activity of IMI@AgNPs@CMSNs.

[0038] (A, B) Comparison of intestinal fluorescence images and intensities of FITC@AgNPs@CMSNs, FITC, and the control group (Student t-test). * P <0.05).

[0039] (C, D) Survival rate and mortality rate of cotton aphids after treatment with IMI or IMI@AgNPs@CMSNs (Student t-test, * P <0.05).

[0040] (E) Pot test apparatus for toxicity bioassay.

[0041] Figure 6 : Toxicity of IMI@AgNPs@CMSNs against ladybugs.

[0042] (A) The mortality rate of ladybugs to IMI and IMI@AgNPs@CMSNs.

[0043] (B) After 50% median lethal dose (LC50) 50 The number of ladybugs surviving 2 days after treatment with a stable dose of IMI or IMI@AgNPs@CMSNs.

[0044] (C) Changes in the number of ladybugs preyed upon exposure to sublethal doses of IMI or IMI@AgNPs@CMSNs.

[0045] (D) Experiment on predation ability designed for ladybugs.

[0046] (EG) Functional response equation of ladybug predators established based on IMI, IMI@AgNPs@CMSNs and control group data.

[0047] Figure 7 The preparation process of IMI@AgNPs@CMSNs and its mechanism of action in enhancing the insecticidal activity of imidacloprid. Detailed Implementation

[0048] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0049] For reference as follows Figures 1-7 The present invention will be described as follows: Cotton aphids were collected from four different cotton-growing regions in China. Aphis gossypii Geographical population samples of *Glover* were collected. Sampling sites were located in Huyanghe City (XJHYH), Jinghe County (XJJH), Hengshui City (HBHS), and Dongying City (SDDY), Shandong Province. The cotton aphid in upland cotton... Gossypiumhirsutum Artificial rearing was carried out on (L.) under experimental conditions of 20-23℃ and 60-70% relative humidity, using a photocycle pattern of 16 h light / 8 h dark.

[0050] Carboxyl-functionalized mesoporous silica nanoparticles (CMSNs) and silver nanoparticles (AgNPs) were purchased from Suzhou Beike Nanotechnology Co., Ltd. (Suzhou, China). Imidacloprid (95.3% industrial grade) was purchased from DuPont Agrochemicals Ltd. (Shanghai, China).

[0051] Morphological observation of samples was performed using a Zeiss GeminiSEM 300 scanning electron microscope (SEM) from Germany. Elemental composition analysis of nanoparticles was obtained using a JED-2300T energy-dispersive spectroscopy (SEMEDS) instrument from NEC Corporation, Japan. Zeta potentials were determined using a Zetasizer Nano ZS90 instrument from Malvern Instruments Ltd., UK, with each sample tested three times (25℃). This instrument was also used to measure the particle size of silver nanoparticles (AgNPs), carbon-metal nanoparticle composites (CMSNs), silver nanoparticle@CMSNs composites, and mesoporous silica@silver nanoparticle@CMSNs composites, with each measurement performed three times (25℃). Fourier transform infrared spectroscopy data were acquired using a Nicoleti S20 instrument from Thermo Fisher Scientific, USA, with a scanning range of 4000-400 / cm, using 64 scans as one complete data point, and a resolution of 2 cm.

[0052] To quantitatively determine the imidacloprid loading of IMI@AgNPs, IMI@CMSNs, and IMI@AgNPs@CMSNs complexes, high-performance liquid chromatography (HPLC) was used. AgNPs powder (5.0 mg), CMSNs powder (5.0 mg), or AgNPs@CMSNs powder (5.0 mg) was dispersed in imidacloprid solutions at concentrations of 60.0, 120.0, 240.0, 480.0, 960.0, 1920.0, and 3840.0 mg / L to prepare IMI@AgNPs, IMI@CMSNs, and IMI@AgNPs@CMSNs complexes, respectively. The imidacloprid in the supernatant was collected for HPLC analysis at a wavelength of 269.0 nm. Analysis was performed using an Agilent 1260 series HPLC system on a Zorbax SB-Aq C18 column (5.0 μm, 4.6 mm × 250.0 mm; Agilent). The mobile phase was a mixture of 75.0% ultrapure water and 25.0% acetonitrile, and the flow rate was 1.0 mL / min. A standard curve for imidacloprid was established to determine its concentration in the supernatant.

[0053] To determine the toxicity of imidacloprid and the IMI@AgNPs@CMSNs complex to cotton aphids, a modified leaf immersion method (Moores et al., 1996) was used. Imidacloprid (95.3% industrial grade) was purchased from DuPont Agrochemicals Ltd. (Shanghai, China). The IMI@AgNPs@CMSNs complex was prepared according to the aforementioned method and serially diluted with distilled water containing 0.05% (v / v) Triton X-100 to adjust the final concentration. Cotton leaf samples with a diameter of 20 mm were immersed in different concentrations of imidacloprid or IMI@AgNPs@CMSNs solutions for 15 s. The control group used an aqueous solution containing 0.05% (v / v) Triton X-100. After treatment, the samples were air-dried on disposable PE gloves and then inverted and spread on the surface of the agar matrix in 12-well cell culture plates. Wingless adult aphids were then precisely transferred to the sample leaves.

[0054] Experimental samples were covered with Chinese art paper to prevent escape. Bioassays were performed in the laboratory at a controlled temperature of 21-23°C with a 16-hour light-8-hour dark cycle. Three replicates were set up for each concentration treatment, with at least 30 aphids per sample. Mortality was assessed 48 hours post-treatment. LC was calculated using POLO Plus 2.0 statistical software (LeOra Software, Berkeley, California, USA) via probabilistic analysis. 50 value.

[0055] The preparation method of AgNPs@CMSNs was as described above. Leaf discs were treated with 1000 mg / L AgNPs@CMSNs for 15 seconds. The treated leaf discs were then air-dried on disposable PE gloves and placed upside down on an agar bed in a 12-well cell culture plate. Wingless adult aphids were carefully transferred to the surface of the discs and covered with Chinese art paper to prevent escape. Wingless adult aphids treated with distilled water served as a control group. Two days after treatment, the aphid gut was collected for 16S rDNA sequencing.

[0056] For 16S rRNA sequence extraction, the sampled aphids were first immersed in 75% ethanol for 90 seconds to remove surface bacteria. The entire intestine was then dissected under a stereomicroscope and directly transferred to centrifuge tubes containing DNA extraction buffer. Eighty intestinal samples were dissected in each replicate experiment, with six replicates for each treatment. After extracting intestinal DNA using a DNA extraction kit (Beijing Qingke Company), high-throughput sequencing of the V4 region of the 16S rRNA gene was performed at Novizan Gene (Beijing), following previous research (Caporaso et al., 2011). The V4 variable region of the 16S rRNA was amplified using primers 505F (5′-CCTAYGGGRBGCASCAG-3) and 806R (5′-GGACTACN NGGGTATCTAA-3).

[0057] Subsequently, the antibacterial activity of silver nanoparticles (AgNPs) and silver nanoparticles loaded with mesoporous carbon nanotubes (AgNPs@CMSNs) against the intestinal commensal bacteria *Sphingomonas* and *Pseudomonas* was investigated by comparing inhibition zones and minimum inhibitory concentrations (MICs). In the inhibition zone test, 1.0 μL of *Sphingomonas* or *Pseudomonas* inoculum with an OD600 value of 0.8 was inoculated onto 20.0 mL 2×YT agar plates with a diameter of 0.3 cm. Then, 60.0 μL of AgNPs and silver nanoparticles loaded with mesoporous carbon nanotubes at concentrations of 0.5 mg / mL, 1.0 mg / mL, 2.0 mg / mL, and 4.0 mg / mL, respectively, were added to the wells. After incubation at 27 ℃ for 48 h, the diameter of the inhibition zone was measured, and each concentration was repeated three times.

[0058] To obtain the MIC values ​​of AgNPs and AgNPs@CMSNs against intestinal commensal bacteria, the bacteria were suspended at a concentration of 1.5 × 10^-6 CFU / .

[0059] After adding 100.0 μL of bacterial solution to each well of a 96-well plate, 100.0 μL of AgNPs or Ag@CMSNs should be added to the bacterial solution. The concentrations of *Streptococcus* and *Pseudomonas* should be measured every 3 hours. Specifically, 1200 mg / L AgNPs and 600 mg / L Ag@CMSNs solutions were prepared as described above, and then serially diluted to the target concentrations. Subsequently, 100 μL of bacterial solution and 100 μL of AgNPs or AgNPs@CMSNs were added to each well. The concentrations of AgNPs were 600.0 mg / L, 300.0 mg / L, 150.0 mg / L, and 75.0 mg / L, respectively, while the concentrations of AgNPs@CMSNs were 300.0 mg / L, 150.0 mg / L, 75.0 mg / L, and 37.5 mg / L, respectively. For each concentration of AgNPs or AgNPs@CMSNs, three replicate wells were set up. 200.0 μL of bacterial solution was added to the wells without AgNPs or AgNPs@CMSNs as a control.

[0060] The degradation capacity of intestinal symbiotic bacteria for imidacloprid was determined by high-performance liquid chromatography (HPLC, 1260 series; Agilent) on a Zorbax SB-Aq C18 column (5.0 μm, 4.6 mm × 250.0 mm; Agilent). 10.0 mL of inoculum containing intestinal symbiotic bacteria with an OD600 of 0.8 was centrifuged at 8000 rpm and then 50.0 mL of liquid mineral medium (MM) (9.5 mM KH₂PO₄, 4.8 mM MgSO₄, 0.1 mM CaCl₂, 0.8 mM Na₂HPO₄) was added, with either 25.0 mg / L or 50.0 mg / L imidacloprid added as the sole carbon source. Degradation experiments were conducted at 28 ℃ and 180 rpm, and the concentrations of imidacloprid and its metabolites were detected by HPLC at a wavelength of 269.0 nm every two days. The mobile phase consisted of 75.0% ultrapure water and 25.0% acetonitrile, with a flow rate of 1.0 mL / min. The concentrations of imidacloprid and its metabolites were calculated based on their standard curves. In this experiment, the standards imidacloprid (DuPont, USA), urea-imidacloprid (Standard Instruments, China), and 5-OH-imidacloprid (Xiyuan Company, China) were all prepared with acetonitrile. Imidacloprid metabolites were determined using an Agilent 1260 / 6520 liquid chromatography-mass spectrometry (LC-MS / MS). Mass spectrometry analysis was performed using an Agilent 1290 LC-MSD in electrospray ionization (ESI) mode, with chromatographic conditions consistent with high-performance liquid chromatography (HPLC). Metabolites were identified by characteristic fragment ions from second-order mass spectrometry and verified by comparison with standards or structural analogs.

[0061] The contact angle characteristics of deionized water, 200.0 mg / L AgNPs, 200.0 mg / L AgNPs@CMSNs, and 200.0 mg / L IMI@AgNPs@CMSNs were characterized on young cotton leaves using an OCA20 instrument (Data-Physics, Germany). Leaf adhesion was assessed using a simulated rain washout test to detect the adhesion effect of IMI@AgNPs@CMSNs. Briefly, 200.0 μL of 200.0 mg / L IMI@CMSNs and 200.0 mg / L IMI@AgNPs@CMSNs were uniformly sprayed onto the surface of young cotton leaves with a diameter of 50.0 mm and allowed to air dry. Subsequently, 1.0 mL of deionized water was uniformly sprayed onto the treated leaves at a flow rate of 0.2 mL / s. Cotton leaves treated with deionized water served as a control group, and each treatment was repeated five times. After 0, 2, and 4 rinses, the distribution of the samples on cotton leaves was further observed using a scanning electron microscope (ZEISS Sigma360, Germany). Residual IMI on the leaves was collected after rinsing with deionized water, and the remaining IMI was recovered with acetonitrile. The IMI recovery rate was determined by HPLC-MS according to the above method to evaluate the adhesion characteristics of IMI, IMI@CMSNs, and IMI@AgNPs@CMSNs.

[0062] 1 mL of FITC was added to 9.0 mL of 500.0 mg / L CMSNs, and the mixture was shaken at 200 rpm for 1 h. The FITC@CMSNs nanoparticles were then collected by centrifugation. Adult aphids were carefully transferred to cotton leaf discs (40.0 mm in diameter) and placed upside down on the agar matrix of 6-well cell culture plates. The aphids were then treated with nanosprays containing either 60.0 μL of 1.0 mg / mL FITC@CMSNs or 1.0 mg / mL FITC, while aphids treated with pure water served as a control group. After 4 h of treatment, the entire gut of the aphids was dissected, and the fluorescence intensity was observed using an inverted fluorescence microscope (Ti-E type, Nikon Corporation, Japan).

[0063] In the greenhouse experiment, three cotton seedlings were cultivated in each pot. Thirty live wingless adult aphids were transferred to each cotton leaf and sprayed with either 50.0 μL of IMI@AgNPs@CMSNs or IMI (both containing 28.7 mg / L IMI). A pure water treatment served as a control group. All treatment groups were sealed with gauze to prevent aphid escape. Each treatment was replicated 16 times, and mortality was assessed daily.

[0064] The insecticidal activity of IMI@AgNPs@CMSNs against ladybugs was evaluated using the insecticide-impregnated filter paper method. Imidacloprid and IMI@AgNPs@CMSNs formulations were prepared according to the above method and serially diluted with distilled water containing 0.05% (v / v) Triton X-100 to adjust the final concentration. Filter paper discs were placed at the bottom of a 9cm diameter, 2cm high petri dish, and 1mL of either imidacloprid or IMI@AgNPs@CMSNs solution was added for treatment. Third-instar ladybug larvae were then transferred to the filter paper discs and fed with sufficient aphids. Mortality was observed and recorded at 24h and 48h after treatment. Ten ladybugs were used for each concentration setting, and the experiment was repeated three times. The LC50 was calculated using the formula. 50 value.

[0065] In this experiment, the concentrations of the test agents imidacloprid and IMI@AgNPs@CMSNs were both 7.0 mg / L, and ladybugs were treated with them for 24 hours. Subsequently, one ladybug was transferred to a petri dish with a diameter of 9 cm and a height of 2 cm, and density gradients of 40, 80, 160, 240, and 320 aphids were set up. Each density setting was repeated six times, with ladybugs treated with distilled water serving as the control group. The theoretical predatory capacity of ladybugs was calculated using the Holling method and the following formula: Na = aN0 / (1 + aT) h N0) Where Na (heads) is the number of predators, N0 (heads) is the prey density, a is the instantaneous detection rate, and T... h This is the duration of the test.

[0066] The formation of IMI@AgNPs@CMSNs indicates that IMI has been successfully loaded onto the AgNPs@CMSNs vector. Figure 1 D). Dynamic light scattering (DLS) data showed that the average particle size of AgNPs was approximately 79.1 nm, while that of CMSNs was 259.9 nm. Furthermore, the particle sizes of AgNPs@CMSNs and IMI@AgNPs@CMSNs increased to 426.2 nm and 546.3 nm, respectively. Figure 1 E). According to zeta potential data, AgNPs and CMSNs exhibit negative charges (−21.6 mV and −15.8 mV, respectively), while the absolute values ​​of AgNPs@CMSN and IMI@AgNPs@CMSNs decrease to −29.2 mV and −38.9 mV, respectively. Figure 1F). Zeta potential is a key indicator for evaluating the stability of colloidal systems; a larger absolute value indicates a more stable colloidal system. The absolute values ​​of the zeta potentials of AgNPs@CMSN and IMI@AgNPs@CMSN are significantly higher than those of AgNPs and CMSNs alone, indicating that their dispersion systems have higher stability compared to single-component systems. Fourier transform infrared spectroscopy (FTIR) analysis further confirmed the characteristic chemical structures of AgNPs@CMSNs, IMI, and IMI@AgNPs@CMSNs. Figure 1 G). As observed in curves I (AgNPs@CMSNs) and III (IMI@AgNPs@CMSNs), it is located at 1101.1 cm. -1 The peak is attributed to the antisymmetric stretching vibration of Si-O-Si, while the peak at 1636.9 cm⁻¹ is... -1 This is a typical characteristic peak of the -carboxyl group. The presence of IMI in IMI@AgNPs@CMSNs was confirmed by comparing the FTIR spectra of pure IMI, AgNPs@CMSNs, and IMI@AgNPs@CMSNs. From the II curve of pure IMI, the peak at 1561.5 cm⁻¹... -1 and 1240.4 cm -1 Stretching vibrations of N−O and C−O were detected at 1435.2 cm⁻¹, while the stretching vibration peaks of -CH₂ and -CH₃ appeared at 1435.2 cm⁻¹, respectively. -1 and 1364.8cm -1 These characteristic peaks appeared in the FTIR spectra of IMI@AgNPs@CMSNs, thus FTIR measurements confirmed the successful encapsulation of IMI in AgNPs@CMSNs. The adsorption capacities of AgNPs, CMSNs, and AgNPs@CMSNs for IMI were analyzed based on the standard curve of IMI. The results showed that at a concentration of 3840.0 mg / L, the maximum adsorption capacity of AgNPs@CMSNs for IMI was 610.2 mg / g; while CMSNs also achieved a maximum adsorption capacity of 680.4 mg / g at the same concentration. Figure 1 H). MSNs have shown high adsorption capacity for IMI in drug delivery applications. Both IMI@CMSNs and IMI@AgNPs@CMSNs exhibited high adsorption capacity for IMI. AgNPs also showed adsorption characteristics for IMI, with a maximum adsorption capacity of 121.3 mg / g at a concentration of 3840.0 mg / L. Figure 1 In addition to their potent antibacterial activity, AgNPs have been shown to improve drug delivery efficiency through electrostatic adsorption.

[0067] The synergistic effect of AgNPs@CMSNs on IMI was observed and presented. Results showed that IMI had a significant LC effect on field populations of XJHYH, XJJH, SDDY, and HBHS. 50 The values ​​were 471.1 mg / L, 82.3 mg / L, 96.7 mg / L, and 73.5 mg / L, respectively. Treatment with AgNPs@CMSNs enhanced the toxicity of IMI to *Brucella aegyptiacus*, and the synergistic ratios of AgNPs@CMSNs with IMI for the XJHYH, XJJH, SDDY, and HBHS populations were 2.8, 1.9, 1.2, and 1.8, respectively. Silver nanoparticles disrupt bacterial cell structure components by releasing Ag+, and this inhibitory effect shows promise in antibiotic resistance research. The combined use of antibiotics and pesticides can enhance pesticide toxicity, and their synergistic effect has advantages in pest and disease control. The insecticidal toxicity of IMI@AgNPs@CMSNs to *Brucella aegyptiacus* was significantly enhanced compared to IMI alone. The LC50 values ​​for the four populations of IMI@AgNPs@CMSNs were... 50 The values ​​were 18.9%, 15.6%, 49.3%, and 37%, respectively. Compared with IMI, the pesticide delivery efficiency of this system decreased by 7%. The microneedle-mediated pesticide delivery system exhibited significant insecticidal effects, and this technology has been successfully applied to pest control.

[0068] The toxicity of different pesticides to cotton aphids.

[0069] a Standard error. Confidence interval. SR efficiency ratio = IMI / (IMI * (IMI * (IMI * (IMI * (IMI * (IMI * (IMI * (IMI * (IMI * (IMI * (IMI) ...) * (IMI * (IMI * (IMI * (I A.gossypii LC 500 Values ​​and IMI@AgNPs@MSNs A. gossypii LC 50 The ratio of values.

[0070] To reveal changes in the gut microbiota of aphids, the microbial community characteristics of aphids exposed to AgNPs@CMSNs were investigated using 16S rRNA analysis. Among the four field sample populations, the genus *Buchnera*, the main symbiotic bacterium of aphids, showed the highest relative abundance at the genus level. Figure 2 A). Principal component analysis (PCA) results showed that the gut symbiotic microbiota of these four field populations exhibited aggregated distribution characteristics. Cotton aphid ( Figure 2 C) Population changes in different treatment groups. Studies showed that treatment with AgNPs@CMSNs did not significantly alter the composition and structure of the cotton aphid's gut microbiota. Analysis using the Shannon index and Chao1 index revealed no significant differences in gut symbiotic diversity and species richness among the four field populations before and after treatment. Figure 2 H, I). Figure 2Heatmap B shows that after treatment with AgNPs@CMSNs, the gut microbiota of oligotrophic bacteria in the four wild populations of cotton aphids ( Stenotrophomonas ) and Sphingosine monocytogenes ( Sphingomonas The relative abundance of both *Oligotrophomonas* and *Sphingosine monocytogenes* showed a decreasing trend. To further investigate the significant inhibitory effect of treatment on gut microbiota at the genus level, the differences between groups were analyzed in detail. The experimental results showed that in these four cotton aphid field populations, the relative abundance of *Oligotrophomonas* and *Sphingosine monocytogenes* was significantly reduced after treatment with *AgNPs@CMSNs*. Figure 2 (D, G). To further analyze the dynamic changes of these two bacteria, researchers collected samples before and after treatment for testing. For example... Figure 2 As shown in J and K, this composite material treatment significantly inhibits the insecticide resistance mediated by these two gut microbiota genera through the degradation of specific compounds, which is an important resistance mechanism in insect pests. Inhibiting toxin-degrading microorganisms enhances the susceptibility of pests to insecticides. Studies have confirmed that *Sphingomonas* in the gut microbiota is responsible for degrading methyl isopropyl methanol (IMI), and inhibiting the growth of *Sphingomonas* increases the susceptibility of cotton aphids to IMI. Therefore, silver nanoparticles@extracellular chemically modified nanoparticles (AgNPs@CMSNs) can effectively target the gut microbiota, significantly inhibiting the abundance of *Sphingomonas*, thereby enhancing the susceptibility of cotton aphids to IMI.

[0071] After treatment with AgNPs@CMSNs, the field populations of cotton aphids XJHYH, XJJH, SDDY, and HBHS showed significantly enhanced susceptibility to imidacloprid. Because AgNPs@CMSNs efficiently target the gut microbiota, the abundance of the gut microbiota *Oligotrophomonas* and *Sphingosine monoclonalis* in these four field populations was significantly reduced. This study found that the gut microbiota *Sphingosine monoclonalis* can mediate cotton aphid resistance to imidacloprid by degrading it. Therefore, the antimicrobial activity of AgNPs@CMSNs against *Oligotrophomonas* and *Sphingosine monoclonalis* was further investigated, and the ability of *Oligotrophomonas* to degrade imidacloprid was also tested.

[0072] Here, *Oligotrophomonas* from the gut microbiota were successfully isolated. Based on the 16S rDNA sequence, the phylogenetic relationships of *Oligotrophomonas* were determined. Figure 3 F). The antibacterial activity of silver nanoparticles (AgNPs) and AgNPs@CMSN *Oligotrophomonas* and *Sphingosine Monoclonal* was detected by inhibition zone assay. Figure 3As shown in A, B, and D, the inhibition zone diameters of AgNPs@CMSNs against Oligotrophozoites and Sphingosine Monoclonalella were significantly larger than those of AgNPs. Further determination of the minimum inhibitory concentration (MIC) of AgNPs@CMSNs against Oligotrophozoites and Sphingosine Monoclonalella revealed that the MICs for AgNPs@CMSNs against Oligotrophozoites and Sphingosine Monoclonalella were 150 and 300 mg / L, respectively. Figure 3 C). The results showed that both AgNPs and AgNPs@CMSNs significantly inhibited *Oligotrophomonas* and *Sphingosine Monoclonal*, and AgNPs@CMSNs showed a more significant inhibitory effect on these two gut microbial genera compared to AgNPs. Silver nanoparticles (AgNPs) are prone to aggregation, which leads to a reduction in specific surface area and decreased antibacterial activity. Nano-silica (MSNs) were used as nanocarriers for silver nanoparticles; these microspheres significantly enhanced the antibacterial effect of AgNPs. Furthermore, AgNPs loaded on the MSN shell enabled the sustained release of silver ions (Ag+) and exhibited excellent antibacterial durability. The metabolic pathway and metabolites of *Oligotrophomonas* on imidacloprid were analyzed using high-performance liquid chromatography-mass spectrometry (HPLC-MS). Figure 3 The mass-to-charge ratios (MCRs) of the molecular ion peaks in the HK range were 275.1, 240.1, 211.1, and 212.1, respectively. According to MCR analysis, the metabolites of imidacloprid by *Oligotrophomonas* were 5-OH IMI, nitroso IMI, guanidino IMI, and urea IMI, in that order. 5-OH IMI originated from the hydroxylation reaction of imidacloprid, while nitroso IMI, guanidino IMI, and urea IMI were nitration metabolites, respectively. This indicates that *Oligotrophomonas* degrades imidacloprid through both hydroxylation and nitration metabolic pathways. Based on the standard curves (Figures S3-S5), the concentrations of imidacloprid and its hydroxylated metabolite 5-OH IMI and nitration metabolite urea IMI were further analyzed to determine the metabolic capacity of *Oligotrophomonas* for imidacloprid. After co-culturing with *Oligotrophomonas* for 8 days, the imidacloprid concentration decreased from 25.0 mg / L to 15.3 mg / L, reaching a maximum metabolic efficiency of 38.8%. Meanwhile, the concentrations of 5-OH IMI and urea IMI increased to approximately 6.4 mg / L and 4.1 mg / L, respectively, reaching their peak values. Figure 3 E). The main metabolic pathways of IMI by bacteria are hydroxylation and nitro reduction. Literature reports that *Pseudomonas* metabolites of IMI include 5-OH IMI, nitrosoIMI, and urea IMI. Isolated oligotrophic bacteria from the gut microbiota were found to be capable of degrading IMI.

[0073] Hydroxylation and nitroreduction metabolic pathways. Detoxifying symbiotics exert their resistance-regulating effects by degrading pesticides, substances that would otherwise be surrogate targets for pest control. When detoxifying symbiotics are absent, the host's sensitivity to pesticides increases significantly. Burkholderia degrades fenitrothion (…). Burkholderia The absence of ) leads to the spotted rimo bug ( Riptortus pedestris The susceptibility to fenitrothion increases. When the gut symbiotic bacteria *Citrobacter* (which degrades trichlorfon)... Citrobacter sp. When the abundance of ) is suppressed, in the oriental fruit fly ( B. dorsalis Similar phenomena (CF-BD) were also observed in [the study]. This demonstrates that AgNPs@CMSNs nanoparticles, by precisely targeting the cotton aphid gut microbiome, effectively inhibit the degradation of IMI by the gut symbiotic bacteria *Oligotrophomonas* and *Sphingosine Monoclonal*, thereby creating a synergistic effect against IMI.

[0074] The wettability and adhesion of pesticides are crucial to the effectiveness of foliar spraying, directly impacting subsequent pesticide utilization. This was determined by measuring the contact angle of cotton leaf surfaces. Figure 4 (A) It was found that the contact angles of IMI and IMI@CMSNs on cotton leaves were approximately 56.5° and 54.7°, respectively. However, the contact angles of the improved AgNPs@CMSNs and IMI@AgNPs@CMSNs decreased to 30.3° and 28.8°, respectively. Figure 4 E). This indicates that nano-silver (AgNPs) plays a key role in improving the wettability of IMI@AgNPs@CMSNs. Solutions with added AgNPs significantly improve surface wettability by reducing the contact angle. To comprehensively evaluate the distribution and adhesion characteristics of IMI@AgNPs@CMSNs on cotton leaf surfaces, a rainwater leaching process was simulated ( Figure 4 A). Figure 4 B and C show the deposition changes of different samples on cotton leaves before and after rainwater leaching. Scanning electron microscopy images show that, with prolonged rinsing time, the residual amount of IMI@AgNPs@CMSNs nanoparticles on the cotton leaves remained slightly higher than that of the IMI@CMSNs-treated group. After simulated rain rinsing, the recoveries of the IMI@AgNPs@CMSNs and IMI@CMSNs groups reached 32.7% and 25.6%, respectively, while the recovery rate of the IMI group dropped to below 4.4%. Figure 4(D) The porous structure allows the nanocarriers to adhere firmly to the leaf surface folds after rainwater runoff, effectively preventing pesticide slippage. Furthermore, the enhanced wettability of IMI@AgNPs@CMSNs significantly strengthens the hydrogen bonds formed with the leaf wax components. This dual effect of mechanical bonding and chemisorption enhances the adhesion between IMI@AgNPs@CMSNs and the leaf surface. Nanoparticle formulations improve pesticide wettability, adhesion, and deposition properties, thereby increasing contact toxicity and enhancing insecticidal activity against target insects. It was found that the improved water permeability, leaf wettability, and adhesion properties significantly enhanced the systemic activity and contact toxicity of the IMI@AgNPs@CMSNs nanocomposite material against cotton aphids.

[0075] Body wall penetration delivery systems are convenient and efficient. Mesoporous silica nanoparticles (MSNs)-mediated pesticide delivery systems have been widely used in pest control. This study used FITC@CMSNs nanoparticles to evaluate the delivery efficiency of CMSNs to compounds within aphids. Figure 5 As shown in A and B, FITC exhibits green fluorescence after penetrating the gut, and the intensity of green fluorescence is significantly enhanced in the gut of aphids treated with FITC@CMSNs. Previous studies have shown that hollow mesoporous silica (RHMS) can effectively improve the permeability of double-stranded RNA and imidacloprid (IMI), thus significantly enhancing the toxicity of the RHMS-based double-stranded RNA / imidacloprid delivery system (2.0-fold increase). This indicates that CMSNs-mediated pesticide delivery systems have advantages in improving pesticide permeability, which is beneficial for targeted pesticide delivery. Therefore, the significantly improved penetration efficiency of IMI@AgNPs@CMSNs enhances the targeted delivery of IMI within aphids, contributing to the potent insecticidal toxicity of IMI@AgNPs@CMSNs against woolly aphids.

[0076] Greenhouse experiments also evaluated the insecticidal activity of IMI@AgNPs@CMSNs against cotton aphids, using potted testing apparatus for toxicological bioassays, such as... Figure 5 As shown in Figure E, the mortality rate of cotton aphids was observed, and the results showed that the control efficacy of the IMI@AgNPs@CMSNs complex against cotton aphids increased by 19.2% within 6 days. Figure 5 (C, D). However, as Figure 6 As shown in Figure A, the toxicity of IMI@AgNPs@CMSNs to ladybugs was significantly reduced compared to IMI. The LC50 of IMI@AgNPs@CMSNs against ladybugs... 50 The value was 144.7 mg / L, which is 2.3 times higher than IMI (62.9 mg / L). Figure 6 B demonstrated via LC 50The survival status of ladybugs after treatment with IMI@AgNPs@CMSNs and IMI was compared with that of the control group. This may be because the application of IMI@AgNPs@CMSNs reduced the amount of IMI exposed to ladybugs. Therefore, IMI@AgNPs@CMSNs enhanced the insecticidal activity against the cotton aphid and inhibited the toxicity against the non-target predator ladybug. The toxicity of different pesticides to ladybugs. ⁿ Standard error. ᵇ Confidence interval. ᶜ SR synergy = LC500 value of IMI@AgNPs@MSNs / ladybug / LC 50 IMI to the beta ratio of ladybugs.

[0077] Researchers also examined the predation capacity of ladybugs treated with sublethal doses of IMI@AgNPs@CMSNs and IMI. When the aphid density increased to 240 aphids / m², the number of ladybugs preyed on in the IMI-treated group was significantly lower than that in the control group. Figure 6 C, D). Further confirmation using the predator-functional response equation indicated that IMI treatment suppressed the predatory ability of ladybugs. Figure 6 E, F). Previous studies have shown that sublethal doses of pesticide exposure can negatively impact predation ability. When cotton bollworms ( Spodoptera frugiperda The density increased to 8 birds / m³ 2 At that time, black spiny whiteflies treated with imidacloprid ( Podisus nigrispinus The predation ability of ladybugs was significantly reduced. It is noteworthy that the sublethal effects of pesticides on non-target organisms (such as inhibiting the predation ability of natural enemies) warrant attention. As shown in the figure, at 6 ℃, the IMI@AgNPs@CMSNs treatment exhibited a slight inhibitory effect on the predation number of ladybugs in high-density aphid populations (240 and 480 individuals). Predator functional response equation analysis indicated that IMI@AgNPs@CMSNs did not significantly reduce the predation ability of ladybugs. Figure 6 (EG). This demonstrates that IMI@AgNPs@CMSNs can effectively mitigate the negative impact of this material on predators, thereby ensuring environmental safety.

[0078] In summary, IMI@AgNPs@CMSNs were successfully prepared. Through the synergistic effect of AgNPs and CMSNs, IMI@AgNps@CMSNs exhibited strong toxicity against the IMI-resistant cotton aphid. The self-assembled AgNPs@CMSNs complex efficiently targets the gut symbiotic flora in cotton aphids that degrade IMI. Subsequently, the AgNPs@CMSNs-mediated IMI delivery system significantly enhanced the insecticidal efficacy of IMI against cotton aphids by inhibiting the detoxification effect of symbiotic bacteria. Compared with IMI alone, the IMI@AgNPs@CMSNs complex exhibited superior wettability, adhesion, and permeability. These performance improvements not only enhanced targeting ability but also effectively inhibited IMI migration, thereby achieving efficient delivery. Notably, the insecticidal effect of IMI@AgNPs@CMSNs against ladybugs decreased compared with IMI alone, and the predatory ability was slightly weakened, indicating that the composite material reduces environmental risk.

[0079] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An insecticide delivery formulation based on binary synergistic nanoparticles, characterized in that: Silver nanoparticles (AgNPs) and carboxyl-functionalized mesoporous silica nanoparticles (CMSNs) were combined to obtain the complex AgNPs@CMSNs.

2. The insecticide delivery formulation based on binary synergistic nanoparticles according to claim 2, characterized in that: The complex AgNPs@CMSNs was combined with imidacloprid to obtain the complex IMI@AgNPs@CMSNs.

3. A method for preparing an insecticide delivery formulation based on binary synergistic nanoparticles, characterized in that, Includes the following steps: Step 1: Dissolve 0.5 mg CMSNs in 5 mL of phosphate-buffered saline (PBS) at pH 7.4 and add 0.5 mg AgNPs; Step 2: Sonicate the mixture for 10 min and then vibrate it at 200 rpm for 4 h; Step 3: Recover AgNPs@CMSNs nanoparticles by centrifugation at 10,000 rpm for 5 min; Step 4: Wash with deionized water and dry under vacuum to obtain the complex AgNPs@CMSNs.

4. The method for preparing an insecticide delivery formulation based on binary synergistic nanoparticles according to claim 3, characterized in that, It also includes the following steps: Step 5: Dissolve imidacloprid in acetone to prepare a mother liquor (10,000 mg / L), then adjust it to 5,000 mg / L with distilled water; Step 6: Suspend 1 mg AgNPs@CMSNs in 10 mL of 5000 mg / L imidacloprid solution, sonicate the mixture for 10 min and shake at 200 rpm for 4 h to obtain the complex IMI@AgNPs@CMSNs.

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