PH / esterase dual-response nano pesticide delivery system as well as preparation method and application thereof

Abstract

The invention discloses a pH / esterase dual-response nano pesticide delivery system as well as a preparation method and application thereof. The nano pesticide delivery system comprises a carboxymethyl chitosan-polycaprolactone graft copolymer (CMCS-g-PCL) carrier, and a pyrethroid pesticide which is loaded on the carrier through physical embedding. The nano pesticide delivery system has good storage stability and foliar affinity, and the photolysis resistance and enzymolysis resistance of the loaded pesticide are remarkably improved. After application, the system shows excellent environmental responsiveness, keeps stable deposition under an acidic condition, and accurately triggers pesticide release in alkaline intestinal tracts of lepidoptera pests and in a carboxylesterase environment. The method is suitable for precise prevention and control of lepidoptera pests, and has the effects of pesticide reduction and environmental risk regulation and control.

Description

Technical Field

[0001] This invention belongs to the field of pesticide formulation technology, specifically relating to a pH / esterase dual-response nanopesticide delivery system, its preparation method and application, which is particularly suitable for intelligent controlled release of pyrethroid pesticides and precise control of lepidopteran pests. Background Technology

[0002] Traditional pesticide formulations such as emulsifiable concentrates and microemulsions suffer from problems such as spray drift, droplet rebound, and photodegradation, resulting in low utilization of active ingredients. While pyrethroid pesticides, represented by lambda-cyhalothrin (LCT), possess broad-spectrum insecticidal efficacy and low mammalian toxicity, long-term overuse has led to widespread metabolic resistance in lepidopteran pests, limiting their long-term effectiveness. Furthermore, compared to traditional insecticides, LCTs pose a higher potential risk to aquatic organisms, presenting a dual challenge to sustainable agricultural development.

[0003] Carboxymethyl chitosan (CMCS), a derivative of chitosan, has attracted much attention in the field of intelligent pesticide delivery due to its advantages such as improved drug solubility, good pH responsiveness, biocompatibility, and biodegradability. However, CMCS faces two major challenges in practical applications: the strong hydrophilicity of its natural polysaccharide backbone leads to insufficient adhesion persistence on plant surfaces, and its single pH response mechanism is difficult to adapt to complex field environments. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention aims to overcome the shortcomings of existing pesticide formulations, such as low utilization rate of active ingredients, poor environmental stability, easy induction of pesticide resistance in pests, and high environmental risks. It provides a pH / esterase dual-response nanopesticide delivery system, its preparation method, and its application.

[0005] In a first aspect, the present invention provides a pH / esterase dual-response nanopesticide delivery system comprising a carboxymethyl chitosan-polycaprolactone graft copolymer (CMCS-g-PCL) carrier and a pyrethroid insecticide loaded on the carrier by physical encapsulation.

[0006] Furthermore, the pyrethroid insecticide is lambda-cyhalothrin (LCT).

[0007] A second aspect of the present invention provides a method for preparing a pH / esterase dual-response nanopesticide delivery system, comprising the following steps:

[0008] 1) Preparation of CMCS-g-PCL support:

[0009] 1.1) Polycaprolactone-carboxylated PCL-COOH and glycidyl methacrylate (GMA) were reacted in 1,4-dioxane with triethylamine as a catalyst and hydroquinone as a polymerization inhibitor at 90-110°C for 5-7 hours under nitrogen protection. After purification, PCLG was obtained. The purification was carried out by removing the solvent by rotary evaporation, filtration and washing with deionized water, and adding cold methanol to precipitate the precipitate.

[0010] 1.2) PCLG was hydroxymethylated via Williamson etherification to generate the etherified product O-PCLG;

[0011] 1.3) Mix the carboxymethyl chitosan CMCS aqueous solution with the 1,4-dioxane solution of O-PCLG, react at 75℃-85℃ for 5h-7h under a nitrogen atmosphere, dialyze, and freeze dry to obtain the graft copolymer CMCS-g-PCL.

[0012] 2) Preparation of nanopesticide delivery systems:

[0013] CMCS-g-PCL was dissolved in water to form an aqueous phase. An ethanol solution of pyrethroid insecticide was added dropwise under vigorous stirring. After the addition was complete, the stirring was switched to low speed for 4-5 hours. Then, the ethanol was removed by dialysis, and the free pyrethroid insecticide was removed by filtering through a membrane to obtain a pH / esterase dual-response nanopesticide delivery system.

[0014] Further, step 1.2) specifically involves: dissolving PCLG in anhydrous dimethylformamide, replacing the air with nitrogen, adding sodium hydride under ice bath conditions, stirring for 1-1.5 hours, slowly adding CH3I, stirring at room temperature for 2-2.5 hours, quenching with ice water, extracting with ethyl acetate, washing with saturated ammonium chloride solution, drying with anhydrous sodium sulfate, and then precipitating with cold methanol after rotary evaporation to obtain the etherified product O-PCLG.

[0015] Further, in step 1.3), the mass ratio is CMCS:O-PCLG = (4:1) - (1:1). Preferably, it is 4:3.

[0016] Further, in step 2), the mass ratio of CMCS-g-PCL:pyrethroid insecticide is (1:1)-(5:1). Preferably, it is 2:1.

[0017] A third aspect of the present invention provides the application of a pH / esterase dual-response nanopesticide delivery system in the control of lepidopteran pests.

[0018] Furthermore, the lepidopteran pest in question is the diamondback moth.

[0019] Further, the method is as follows: the pH / esterase dual-response nanopesticide delivery system is sprayed onto the leaves of crops.

[0020] The beneficial effects of this invention are as follows:

[0021] 1. The nanodelivery system provided by the present invention has pH / esterase dual response characteristics. It specifically hydrolyzes in the alkaline environment (pH=9.0-10.5) of the midgut of lepidopteran pests and in the environment of carboxylesterase overexpression, triggering targeted release; while it is stably deposited under acidic conditions.

[0022] 2. The CMCS-g-PCL vector provided by this invention has a bridged ester bond in its structure that can act as a competitive substrate to bind to the active site of carboxylesterase, thereby improving the resistance of LCT to enzymatic degradation and effectively delaying the development of pesticide resistance in pests.

[0023] 3. The nano-delivery system provided by the present invention uses glycidyl methacrylate as a bridging agent to connect carboxymethyl chitosan and polycaprolactone to form an amphiphilic graft copolymer, which is used for the intelligent delivery of pyrethroid pesticides. It has good storage stability, anti-photolysis properties, enhanced leaf deposition efficiency, and reduced loss of active substances.

[0024] 4. The nanodelivery system provided by this invention significantly improves the control effect on lepidopteran pests. At the same time, due to the intelligent release, it reduces non-target exposure and has lower ecotoxicity to non-target organisms.

[0025] In summary, the nanodelivery system provided by this invention, through a structural modification strategy, constructs a pH / esterase dual-responsive nanopesticide delivery system based on carboxymethyl chitosan-polycaprolactone graft copolymer (CMCS-g-PCL), which improves the deposition stability and resistance to environmental degradation of pesticides on the leaf surface, achieves intelligent targeted release, delays the development of pesticide resistance in pests, and ultimately achieves the dual goals of reducing pesticide application while increasing efficiency and regulating ecological risks. Attached Figure Description

[0026] Figure 1 Transmission electron microscopy image of LCT@CMCS-g-PCL prepared in Example 1 under a weakly acidic environment.

[0027] Figure 2 Storage stability of LCT@CMCS-g-PCL prepared in Example 1.

[0028] Figure 3 The photostability results of LCT@CMCS-g-PCL and LCT prepared in Example 1 are shown.

[0029] Figure 4 The results of anti-enzymatic degradation of LCT@CMCS-g-PCL and LCT prepared in Example 1 are shown.

[0030] Figure 5 The amount of different formulations deposited on cabbage and cucumber leaves in Example 1.

[0031] Figure 6 The cumulative release rate of LCT@CMCS-g-PCL in different release media in Example 1 is given.

[0032] Figure 7 The relative mortality rate of third-instar larvae of diamondback moth after 24h and 48h of treatment with different concentrations of LCT in Example 1. Detailed Implementation

[0033] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention.

[0034] Example 1: A pH / esterase dual-response nanopesticide delivery system (LCT@CMCS-g-PCL)

[0035] (I) The preparation method is as follows:

[0036] 1. Preparation of CMCS-g-PCL support

[0037] 1.1) Preparation of PCLG intermediates

[0038] 1 g of polycaprolactone-carboxyl (PCL-COOH) and 1.5 mL of glycidyl methacrylate (GMA) were dissolved in 20 mL of 1,4-dioxane. 1 wt% of triethylamine catalyst and 0.5 wt% hydroquinone polymerization inhibitor were added. The mixture was stirred at 100 °C for 6 h under a nitrogen atmosphere. After the reaction was completed, the solvent was removed by rotary evaporation. The mixture was washed three times with deionized water and then cooled with methanol to precipitate a white precipitate, yielding the intermediate product PCLG.

[0039] 1.2) Preparation of the etherified product O-PCLG

[0040] The hydroxymethylation of PCLG was performed via Williamson etherification, specifically as follows:

[0041] 1 g PCLG was dissolved in 20 mL of anhydrous dimethylformamide, the air was replaced with nitrogen, 120 mg of sodium hydride was added under ice bath conditions, and the mixture was stirred for 1 h. 500 μL of CH3I was slowly added dropwise, and the mixture was stirred at room temperature for 2 h. After quenching with ice water, extraction with ethyl acetate, washing with saturated ammonium chloride solution, and drying with anhydrous sodium sulfate, the product was precipitated by rotary evaporation and then by cold methanol to obtain the etherified product O-PCLG.

[0042] 1.3) Preparation of graft copolymer (CMCS-g-PCL)

[0043] 0.4 g of CMCS was added to 50 mL of deionized water and stirred until dissolved. Simultaneously, 0.3 g of O-PCLG was dissolved in 3 mL of 1,4-dioxane solution, with a CMCS to O-PCLG mass ratio of 4:3. The O-PCLG solution was added dropwise to the CMCS solution, and the mixture was stirred continuously at 80 °C for 6 h under a nitrogen atmosphere. After the reaction was complete, the resulting reaction solution was dialyzed through a dialysis bag (molecular weight cutoff 8000 Da-14000 Da) for 48 h, and then freeze-dried to obtain the graft copolymer (CMCS-g-PCL).

[0044] 2. Preparation of nanopesticide delivery system (LCT@CMCS-g-PCL)

[0045] 50 mg of CMCS-g-PCL was dissolved in 50 mL of water to form an aqueous phase. Under vigorous stirring, 2.5 mL of LCT ethanol solution with a concentration of 10 mg / mL was added dropwise. The mass ratio of CMCS-g-PCL to LCT was 2:1. After the addition was complete, the mixture was stirred at low speed for 4 h. Then, the ethanol was removed by dialysis, and the free LCT was removed by passing the solution through a 0.8 μm filter membrane to obtain drug-loaded nanoparticles LCT@CMCS-g-PCL. The nanoparticles were then freeze-dried and stored.

[0046] (II) Performance Testing

[0047] 1. Morphological observation of LCT@CMCS-g-PCL nanoparticles

[0048] Figure 1 Transmission electron microscopy (TEM) images of LCT@CMCS-g-PCL nanoparticles in a weakly acidic environment. Figure 1 As can be seen, LCT@CMCS-g-PCL exhibits a spherical structure with uniform size.

[0049] 2. Storage stability of LCT@CMCS-g-PCL nanoparticles

[0050] Figure 2 Results show the long-term storage stability of LCT@CMCS-g-PCL nanoparticles. (From...) Figure 2 It can be seen that after storage at 4±2℃ for 3 months, the particle size of LCT@CMCS-g-PCL nanoparticles was 305.0±2.6nm and the encapsulation efficiency was 60.19±1.64%. There were no significant changes in the particle size and encapsulation efficiency of LCT@CMCS-g-PCL nanoparticles, indicating that the formulation has good storage stability.

[0051] 3. Photodegradation resistance of LCT@CMCS-g-PCL nanoparticles

[0052] Photostability results are as follows Figure 3As shown, under continuous irradiation for 72 hours, the residual rate of LCT in LCT@CMCS-g-PCL was 73.35±2.41%, while the residual rate of free LCT rapidly decreased to 28.11±1.57%. In addition, the half-life of LCT loaded in CMCS-g-PCL was extended from 37.5 hours to 187.9 hours, which slowed down the photodegradation process of LCT.

[0053] 4. Anti-enzyme degradation properties of LCT@CMCS-g-PCL nanoparticles

[0054] The results of enzyme degradation are as follows Figure 4 As shown, the degradation rate of LCT@CMCS-g-PCL nanoparticles under the action of esterases is lower than that of LCT raw material. After loading, the degradation rate of LCT decreased from 41.36% to 19.36% in 24 hours, and the difference in degradation rate became increasingly larger over time.

[0055] 5. Leaf deposition efficiency of LCT@CMCS-g-PCL nanoparticles

[0056] A 2cm diameter leaf sample was fixed on a 45° slope, and 0.5mL of test solution (containing 200μg / mL LCT) was sprayed onto the leaf. Figure 5 The deposition rates of different formulations on cabbage and cucumber leaves are shown. On cabbage leaves, the deposition rate of LCT@CMCS-g-PCL (58.48±6.56%) was significantly higher than that of LCT aqueous dispersion (11.08±2.66%) and commercially available LCT microemulsion (LCT ME, 24.77±9.46%). The amphiphilic structure of LCT@CMCS-g-PCL anchors the waxy layer through PCL segments, delaying the migration of active ingredients during drying. On cucumber leaves, the deposition rate of LCT@CMCS-g-PCL (64.01±3.37%) was also significantly higher than that of LCT aqueous dispersion (19.7±3.87%) and LCT ME (46.95±4.20%), indicating that the hydrophilic surface of the copolymer can achieve efficient deposition through hydrogen bonding and other interactions.

[0057] 6. In vitro release behavior of LCT@CMCS-g-PCL nanoparticles

[0058] Figure 6The cumulative release rate of LCT@CMCS-g-PCL in different release media is shown. At pH 5, LCT release was significantly inhibited, with almost no release after 12 hours and a cumulative release rate of only about 34% after 72 hours. In media containing pH 7 and 9, LCT was rapidly released from CMCS-g-PCL, with over 60% released after 24 hours and approximately 80% cumulatively released after 72 hours. The addition of esterase further enhanced the controlled release capability. In esterase-containing media, the release efficiency was significantly improved under pH 7 and 9 conditions, with almost complete LCT release after 72 hours. This indicates that the esterase hydrolyzed the ester bonds of CMCS-g-PCL, disrupting the hydrophobic core of the material and accelerating drug diffusion. In contrast, the release increase in the esterase-containing medium at pH 5 was limited, with approximately 41% released after 72 hours.

[0059] 7. Bioactivity of LCT@CMCS-g-PCL nanoparticles

[0060] Figure 7 The relative mortality rates of third-instar larvae of the diamondback moth after 24 h and 48 h of treatment with different concentrations of LCT were determined. Compared with the commercial formulation (LCTME), the 48 h LC50 mortality rate of the LCT@CMCS-g-PCL group was significantly lower. 50 The value decreased by 39.7%, and its narrower confidence interval indicates a more stable dose-response relationship. LCT@CMCS-g-PCL exhibited higher insecticidal efficacy and sustained bioactivity. Example 2: A pH / esterase dual-response nanopesticide delivery system (LCT@CMCS-g-PCL)

[0061] (I) The preparation method is as follows:

[0062] 1. Preparation of CMCS-g-PCL support:

[0063] 1.1) Preparation of PCLG intermediate: Same as in Example 1.

[0064] 1.2) Preparation of the etherified product O-PCLG: Same as in Example 1.

[0065] 1.3) Preparation of graft copolymer (CMCS-g-PCL)

[0066] 0.4 g of CMCS was added to 50 mL of deionized water and stirred until dissolved. Simultaneously, 0.2 g of O-PCLG was dissolved in 2 mL of 1,4-dioxane solution, with a CMCS to O-PCLG mass ratio of 2:1. The O-PCLG solution was added dropwise to the CMCS solution, and the mixture was stirred continuously at 80 °C for 6 h under a nitrogen atmosphere. After the reaction was complete, the resulting reaction solution was dialyzed through a dialysis bag (molecular weight cutoff 8000 Da-14000 Da) for 48 h, and then freeze-dried to obtain the graft copolymer CMCS-g-PCL.

[0067] 2. Preparation of nanopesticide delivery system (LCT@CMCS-g-PCL)

[0068] 50 mg of CMCS-g-PCL was dissolved in 50 mL of water to form an aqueous phase. Under vigorous stirring, 2.5 mL of LCT ethanol solution with a concentration of 10 mg / mL was added dropwise. The mass ratio of CMCS-g-PCL to LCT was 2:1. After the addition was complete, the mixture was stirred at low speed for 4 h. Then, the ethanol was removed by dialysis, and the free LCT was removed by passing the solution through a 0.8 μm filter membrane to obtain drug-loaded nanoparticles LCT@CMCS-g-PCL. The nanoparticles were then freeze-dried and stored.

[0069] Example 3: A pH / esterase dual-response nanopesticide delivery system (LCT@CMCS-g-PCL)

[0070] (I) The preparation method is as follows:

[0071] 1. Preparation of CMCS-g-PCL support:

[0072] 1.1) Preparation of PCLG intermediate: Same as in Example 1.

[0073] 1.2) Preparation of the etherified product O-PCLG: Same as in Example 1.

[0074] 1.3) Preparation of graft copolymer (CMCS-g-PCL)

[0075] 0.4 g of CMCS was added to 50 mL of deionized water and stirred until dissolved. Simultaneously, 0.3 g of O-PCLG was dissolved in 3 mL of 1,4-dioxane solution, with a CMCS to O-PCLG mass ratio of 4:3. The O-PCLG solution was added dropwise to the CMCS solution, and the mixture was stirred continuously at 80 °C for 6 h under a nitrogen atmosphere. After the reaction was complete, the resulting reaction solution was dialyzed through a dialysis bag (molecular weight cutoff 8000 Da-14000 Da) for 48 h, and then freeze-dried to obtain the graft copolymer CMCS-g-PCL.

[0076] 2. Preparation of nanopesticide delivery system (LCT@CMCS-g-PCL)

[0077] 50 mg of CMCS-g-PCL was dissolved in 50 mL of water to form an aqueous phase. Under vigorous stirring, 5 mL of LCT ethanol solution with a concentration of 10 mg / mL was added dropwise. The mass ratio of CMCS-g-PCL to LCT was 1:1. After the addition was complete, the mixture was stirred at low speed for 4 h. Then, the ethanol was removed by dialysis, and the free LCT was removed by passing the solution through a 0.8 μm filter membrane to obtain drug-loaded nanoparticles LCT@CMCS-g-PCL. The nanoparticles were then freeze-dried and stored.

Claims

1. A pH / esterase dual-response nanopesticide delivery system, characterized in that, It includes a carboxymethyl chitosan-polycaprolactone graft copolymer CMCS-g-PCL carrier, and a pyrethroid insecticide loaded onto the carrier by physical encapsulation.

2. The pH / esterase dual-response nanopesticide delivery system according to claim 1, characterized in that, The pyrethroid insecticide is lambda-cyhalothrin (LCT).

3. The preparation method of the pH / esterase dual-response nanopesticide delivery system according to claim 1 or 2, characterized in that, The preparation method includes the following steps: 1) Preparation of CMCS-g-PCL support: 1.1) Polycaprolactone-carboxylated PCL-COOH and glycidyl methacrylate GMA were reacted in 1,4-dioxane with triethylamine as a catalyst and hydroquinone as a polymerization inhibitor under nitrogen protection at 90℃-110℃ for 5h-7h. After purification, PCLG was obtained. 1.2) PCLG was hydroxymethylated via Williamson etherification to generate the etherified product O-PCLG; 1.3) Mix the carboxymethyl chitosan (CMCS) aqueous solution with the 1,4-dioxane solution of O-PCLG under a nitrogen atmosphere. The reaction was carried out at 75℃-85℃ for 5-7 hours, followed by dialyzing and freeze-drying to obtain the graft copolymer CMCS-g-PCL. 2) Preparation of nanopesticide delivery systems: CMCS-g-PCL was dissolved in water to form an aqueous phase. An ethanol solution of pyrethroid insecticide was added dropwise under vigorous stirring. After the addition was complete, the stirring was switched to low speed for 4-5 hours. Then, the ethanol was removed by dialysis, and the free pyrethroid insecticide was removed by filtering through a membrane to obtain a pH / esterase dual-response nanopesticide delivery system.

4. The preparation method of a pH / esterase dual-response nanopesticide delivery system according to claim 3, characterized in that, Step 1.2) Specifically, PCLG is dissolved in anhydrous dimethylformamide, the air is replaced with nitrogen, sodium hydride is added under ice bath conditions, and the mixture is stirred for 1-1.5 hours. CH3I is slowly added dropwise, and the mixture is stirred at room temperature for 2-2.5 hours. After quenching with ice water, extraction with ethyl acetate, washing with saturated ammonium chloride solution, and drying with anhydrous sodium sulfate, the product is precipitated by rotary evaporation and then by cold methanol to obtain the etherified product O-PCLG.

5. The preparation method of a pH / esterase dual-response nanopesticide delivery system according to claim 3, characterized in that, In step 1.3), the mass ratio is CMCS:O-PCLG=(4:1)-(1:1).

6. The preparation method of a pH / esterase dual-response nanopesticide delivery system according to claim 3, characterized in that, In step 2), the mass ratio of CMCS-g-PCL:pyrethroid insecticide is (1:1)-(5:1).

7. The application of the pH / esterase dual-response nanopesticide delivery system according to claim 1 or 2 in the control of lepidopteran pests.

8. The application according to claim 7, characterized in that, The lepidopteran pest in question is the diamondback moth.

9. The application according to claim 8, characterized in that, The method is as follows: the pH / esterase dual-response nanopesticide delivery system is sprayed onto the leaves of crops.

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