Preparation and application of functionalized nanometal drug-loaded particles for seed treatment

Abstract

The application discloses a preparation method of nano metal drug-loaded particles for seed treatment and application of the nano metal drug-loaded particles in prevention and treatment of rice diseases and pests. The nano metal drug-loaded particles are prepared by using an organic metal framework to prepare a nano carrier and load pesticides, and an assembly tannic acid metal complex shell is used to perform targeted controlled release of the pesticides at a harmful organism damage site, so that the nano metal drug-loaded particles can effectively cope with massive reproduction of pathogenic bacteria under acidic conditions and damage of pests. The nano metal drug-loaded particles have excellent targeted harmful organism prevention and treatment effect, improve effective utilization of active ingredients of the pesticides, prolong a holding period, promote germination of rice seeds and growth of seedlings, are particularly suitable for improving seedling quality in rice direct seeding cultivation, and have wide application prospects in rice seed treatment.

Description

Technical Field

[0001] This invention relates to the field of pesticide formulation processing and application, specifically to the preparation of nano-pesticides for rice seed treatment, the prevention and control of rice seedling diseases and pests, and the improvement of seedling quality. This invention provides nano-pesticide treatment technology for rice seeds, suitable for direct seeding and efficient control of seedling diseases and pests, thus simplifying rice cultivation. Background Technology

[0002] Rice is a crucial staple crop in my country, and the health of its seedlings is vital to rice yield. The rice seedling stage is susceptible to various harmful organisms, including seed-borne and soil-borne diseases and pests, making it a critical period for pest and disease control. In recent years, the continuous development of direct seeding and drone seeding technologies has provided significant opportunities for simplified cultivation. Direct-seeded rice often suffers from weak buds, uneven emergence, shallow roots, and lodging, necessitating the development of related seed treatment technologies and products. Treating seeds with pesticides can effectively reduce pest and disease damage during the seedling stage. Seed treatments such as seed coating, seed dressing, seed soaking, and seed pelleting allow for concealed application, saving labor and effort. However, currently, domestically produced seed coating agents are mainly suspension concentrates, with limited formulations and poor inhibitory effects on pathogens residing within the seeds, leading to serious pesticide resistance. Furthermore, pesticide damage caused by seed soaking is also common. Therefore, the development of highly effective and safe pesticide formulations that promote seedling growth is key to solving these problems.

[0003] Nanomaterials, with their small particle size and large specific surface area, possess strong capabilities for targeted transport and translocation of pesticides to harmful organisms. They enable the on-demand release of pesticide active ingredients or release in response to various stimuli (pH, temperature, humidity, light, etc.) at specific times and spaces, providing an effective way to achieve efficient pesticide control of harmful organisms while ensuring environmental and non-target organism safety. Furthermore, some nanomaterials exhibit bactericidal and disease-preventing effects, promote plant growth, and improve crop biomass and fruit quality. However, the application of nanopesticides in seed treatment is rarely reported, particularly regarding nanocarriers and formulations with functions such as pest control, seed priming, and seed vigor enhancement. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing functionalized nano-metallic drug-loaded particles for seed treatment. This nano-pesticide is used for intelligent and efficient control of plant diseases and pests, has a long-lasting effect, is safe for seedlings, can promote the germination of rice seeds and the growth of rice seedlings, and has high safety for non-target organisms.

[0005] The objective of this invention is achieved as follows:

[0006] In a first aspect, the present invention provides a nano-metal framework as a carrier, wherein a method for preparing the nano-metal framework carrier includes the following steps:

[0007] 1) Dissolve zirconium tetrachloride in N,N-dimethylformamide, and then add terephthalic acid to obtain a mixture;

[0008] 2) Add glacial acetic acid to the mixture obtained in step 1) and carry out a hydrothermal reaction to prepare a nano-metal framework carrier;

[0009] In the method, in steps 1) and 2), the mass ratio of zirconium tetrachloride, terephthalic acid and glacial acetic acid is (1-2):1:(20-40);

[0010] In a preferred embodiment of the present invention, in step 1), zirconium tetrachloride (35 mg), terephthalic acid (25 mg), and N,N-dimethylformamide (10 mL) are mixed and then dissolved by ultrasonication.

[0011] In step 2), the mixture and glacial acetic acid (750 mg) are subjected to a hydrothermal reaction in an oven for 24 h to prepare a nano-metal framework carrier;

[0012] Furthermore, in a second aspect, the present invention provides functionalized metal nanoparticles for drug delivery, said functionalized metal nanoparticles for drug delivery prepared by the following method:

[0013] 1) Dissolve the active ingredient of pesticide in N,N-dimethylformamide, add the nano-metal framework carrier, sonicate and stir at room temperature to adsorb, and obtain nano-metal drug-loaded particles.

[0014] 2) The nano-metal drug-loaded particles obtained in step 1) are modified with tannic acid and metal salts to prepare functionalized nano-metal drug-loaded particles.

[0015] In step 2), the mass ratio of the nano-metal drug-loaded particles, tannic acid, and metal salt is 1:1:(2-4).

[0016] In step 2) above, the method of modification using tannic acid and metal salt as modifiers includes the following steps:

[0017] A) The pesticide-loaded nano-metal framework carrier and surfactant solution were mixed at a mass ratio of 1:1000 and ultrasonically dispersed at room temperature to prepare a nano-metal-loaded drug particle dispersion.

[0018] B) Add tannic acid aqueous solution to the nano-metal drug-loaded particle dispersion obtained in step A), and add metal salt aqueous solution dropwise. After stirring at 1200 rpm for 5 min, vortex for 30 min, centrifuge to obtain precipitate, and wash three times with ethanol to remove unreacted tannic acid and metal salt to obtain functionalized nano-metal drug-loaded particles.

[0019] Preferably, in step A), the ultrasonic time is 60 min, and the mass percentage concentration of the surfactant solution is 0.1-6%, preferably 1%.

[0020] In step A), the surfactant is OP-10#, 4917#, OP-21#, 1086#, 602# and 3300#, preferably 602#;

[0021] In step B), the metal salt is copper sulfate, zinc acetate, or ferric sulfate, preferably zinc acetate.

[0022] In one embodiment of the present invention, the nano-metal-loaded pesticide particles with tebuconazole or fipronil as active ingredients have a pesticide active ingredient to nano-metal skeleton carrier mass ratio of 1:2.

[0023] The advantages of this invention are:

[0024] 1) The functionalized nano-metal drug-carrying particles can be further prepared into nano-powders, nano-water-dispersible granules, nano-particles and nano-suspensions for corresponding seed treatments.

[0025] 2) Compared with the one-pot stirring method, the functionalized nano-metal drug-loaded particles prepared by the present invention have a stable drug loading capacity, which is more in line with the needs of industrial production.

[0026] 3) The functionalized nanoparticles loaded with pesticides obtained by the above loading method have a pesticide loading capacity of 10.55±0.48%. Compared with the original metal-organic framework (MOF) method, the open embedding of pesticides is transformed into closed encapsulation, which significantly improves the controlled release effect of pesticides in response to pH and temperature. This not only increases the pesticide loading capacity of the metal framework but also avoids waste and increased costs of carriers in production, making the application of this nanocarrier in the field of pesticides possible. In addition, it also achieves effective control of pathogens that can be controlled in large quantities under acidic conditions, and improves seed vigor and seedling quality by inducing the breaking of seed dormancy.

[0027] 4) The median diameter of the functionalized nanoparticles prepared by the above method is 142.7 nm. This nanopesticide exhibits good controlled-release performance in the responsive environment, which is beneficial for improving pesticide utilization, precise control, and extending the duration of effectiveness, and has broad prospects for development and application.

[0028] 5) Metal-organic frameworks (MOFs), as porous nanomaterials, achieve diverse structures through the selection of different metal ions and organic ligands. They also possess inherent advantages such as high porosity, large specific surface area, and structural stability. When pesticide nanoparticles are loaded with pesticides using MOFs, a pesticide coating forms on the seed surface during seed treatment. These MOF particles can effectively concentrate nutrients, continuously providing them during seed germination and growth, and promoting nutrient absorption. Furthermore, this structure exhibits antibacterial properties, preventing damage to seeds and seedlings from soil pathogens and pests, thus providing disease and pest control. Plant seeds treated with pesticide-loaded MOF particles are less susceptible to harmful organisms during storage, facilitating long-term preservation and transportation, and possess significant economic value. Attached Figure Description

[0029] Figure 1 The microstructure (AC) and particle size distribution (D) of the nano-metal framework carriers prepared by mass ratios of zirconium tetrachloride, terephthalic acid and glacial acetic acid (1:1:20, 1.4:1:30 and 2:1:40) under scanning electron microscopy.

[0030] Figure 2 The microstructure of functionalized metal drug-loaded nanoparticles prepared using surfactants OP-10# (A; A-1, A-2, A-3), 4917# (B; B-1, B-2, B-3), OP-21# (C; C-1, C-2, C-3), 1086# (D; D-1, D-2, D-3), 602# (E; E-1, E-2, E-3), and 3300# (F; F-1, F-2, F-3) with mass fractions of 0.1%, 1%, and 6% were examined under a scanning electron microscope.

[0031] Figure 3 Microscopic morphology of functionalized metal nanoparticles under scanning electron microscopy (A) and transmission electron microscopy (B), and particle size distribution of functionalized metal nanoparticles (C).

[0032] Figure 4 Infrared spectrum (A), X-ray photoelectron spectrum (B), thermogravimetric analysis (C), transmission electron microscopy combined with energy dispersive spectroscopy (EDS) (D), chlorine element energy dispersive spectroscopy (E), and zinc element energy dispersive spectroscopy (F) of functionalized metal nanoparticles.

[0033] Figure 5 Release curves of nano-metal drug-loaded particles (A, D) and functionalized nano-metal drug-loaded particles (B, E) in solutions at a series of temperatures (10℃, 20℃ and 30℃) and pH (pH=5, pH=7 and pH=9), and schematic diagrams of the release curves of functionalized nano-metal drug-loaded particles fitted by the Peppas model (C, F).

[0034] Figure 6 The inhibitory effects of styraxazole technical, styraxazole suspension, and functionalized nano-metal drug-loaded particles on the in vitro mycelial growth of *Fusarium oxysporum* at different concentrations of styraxazole active ingredient were investigated. The inhibitory effects of functionalized nano-metal drug-loaded particles on the in vitro mycelial growth of *Fusarium oxysporum* under different pH conditions were also studied.

[0035] Figure 7 The inhibitory effect of different concentrations of functionalized nano-metal empty particles (without added pesticide active ingredients) on the growth of Fusarium oxysporum mycelium in vitro.

[0036] Figure 8 The effects of tebuconazole on the safety of rice germination (A) and emergence (B). a, b, and c represent low, medium, and high dose treatments of tebuconazole suspension, respectively; d, e, and f represent low, medium, and high dose treatments of functionalized nano-metal drug-loaded particles, respectively; and g is the blank control.

[0037] Figure 9 The effects of tebuconazole suspension and functionalized nano-metal drug-loaded particles on root length (A), leaf length (B), and fresh weight (C) of rice under low, medium, and high dosage treatments. Detailed Implementation

[0038] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.

[0039] In the following embodiments, the term "parts by mass" is understood in the art to be a unit of weight, such as "g", "kg", "mg", "μg", etc.

[0040] The reagents involved in the following embodiments and test examples are disclosed as follows:

[0041] Zirconium tetrachloride (98%, Beijing Bailingwei Technology Co., Ltd.), terephthalic acid (98%, from Shanghai Yuanye Biotechnology Co., Ltd.), N,N-dimethylformamide (99%, Tianjin Xins Biochemical Technology Co., Ltd.), glacial acetic acid (99%, Tianjin Xins Biochemical Technology Co., Ltd.), tannic acid (96%, Tianjin Xins Biochemical Technology Co., Ltd.), zinc acetate (98%, Shanghai Maclean Biochemical Technology Co., Ltd.), copper sulfate (99%, Shanghai Maclean Biochemical Technology Co., Ltd.), ferric sulfate (97%, Tianjin Xins Biochemical Technology Co., Ltd.), tebuconazole technical grade (98%, Lanxess GmbH, Germany), dinotefuran (99%, Shandong United Pesticide Industry Co., Ltd.), tebuconazole seed treatment suspension (10%, Beinong (Haili) Zhuozhou Seed Coating Co., Ltd.), dinotefuran seed treatment dispersible powder (22%, Guangxi Tianyuan Biochemical Co., Ltd.).

[0042] The rice seed is Longgeng 31, a japonica conventional rice variety.

[0043] The strain of Fusarium fujikura HLB-19, the pathogen of rice seedling blight, was preserved in our laboratory.

[0044] The following uses tebuconazole and dinotefuran as examples to illustrate the preparation method and optimal conditions of the functionalized nano-metal pesticide-loaded particles of the present invention. Those skilled in the art should understand that the pesticides that can be loaded into the functionalized nano-metal pesticide-loaded particles of the present invention include, but are not limited to, tebuconazole and dinotefuran.

[0045] Example 1: Preparation of Nanoscale Metal Framework Carriers

[0046] I. The preparation method of the nano-metal framework carrier consists of the following steps:

[0047] 1) Dissolve zirconium tetrachloride (25 mg, 35 mg, 50 mg) and terephthalic acid (25 mg) in 10 mL of N,N-dimethylformamide under ultrasonic treatment;

[0048] 2) Glacial acetic acid (500 mg, 750 mg, and 1000 mg) was added to the mixture in step 1), i.e., the mass ratios of zirconium tetrachloride, terephthalic acid, and glacial acetic acid were 1:1:20, 1.4:1:30, and 2:1:40, respectively. The mixture was subjected to a hydrothermal reaction in an oven at 120 °C for 24 h to synthesize a metal framework. The precipitate was obtained by centrifugation at 8000 rpm, washed three times with N,N-dimethylformamide to remove residual reactants, and dried in an oven at 60 °C to obtain nano-metal framework carrier particles.

[0049] II. Electron Microscopy Observation Methods

[0050] The nano-metal framework carrier particles were fixed onto the sample holder with conductive adhesive, sputtered with gold, and then placed on the scanning electron microscope sample holder. The sample holder was then evacuated for observation, and the test pressure was 2kV.

[0051] III. Electron Microscopy Observation Results: Scanning electron microscopy revealed that the white powder obtained in steps 1) and 2) exhibited a regular octahedral three-dimensional structure. Figure 1 The mass ratios of zirconium tetrachloride, terephthalic acid, and glacial acetic acid (1:1:20, 1.4:1:30, and 2:1:40) have a slight influence on the morphology of the nano-metal framework carrier. Based on the morphological characteristics of the nano-metal framework carrier, a mass ratio of zirconium tetrachloride, terephthalic acid, and glacial acetic acid of 1.4:1:30 was selected to prepare nano-metal framework carrier particles for subsequent experiments. The median diameter D of the nano-metal framework carrier particles was determined to be... 50 84.7nm ( Figure 1 D).

[0052] Example 2: Preparation and Optimization of Functionalized Metallic Drug-Loaded Nanoparticles

[0053] I. The preparation of functionalized metal nanoparticles using the nano-metal framework carrier obtained in Example 1 includes the following steps:

[0054] 1) Pesticide loading: 2.4 g of tebuconazole or fipronil was dissolved in N,N-dimethylformamide (15 mL), and 4.8 g of the nano-metal framework carrier prepared in Example 1 was added. The mixture was stirred and adsorbed at room temperature. After loading, the mixture was centrifuged and the precipitate was washed. The mixture was then dried in an oven at 60 °C for 3 h to obtain pesticide-loaded nano-metal particles.

[0055] 2) Preparation of functionalized nano-metal drug-loaded particles: A) Nano-metal drug-loaded particles loaded with pesticides and surfactant solution were mixed at a mass ratio of 1:1000 and dispersed thoroughly by ultrasound at room temperature to prepare a nano-metal drug-loaded particle dispersion; B) Tannic acid aqueous solution was added to the nano-metal drug-loaded particle dispersion obtained in step A), and metal salt aqueous solution was added dropwise. After stirring at 1200 rpm for 5 min, the mixture was vortexed for 30 min to deposit a tannic acid metal complex shell on the surface of the metal drug-loaded particles. The precipitate was collected after centrifugation at 8000 rpm, washed three times with ethanol to remove unreacted tannic acid and metal salt, and dried in an oven at 60℃ for 5 h to obtain functionalized nano-metal drug-loaded particles.

[0056] II. Effect of Metal Salt Type on the Reaction Yield of Tannic Acid Metal Complexes

[0057] 1) Test methods

[0058] Tannic acid (0.2g) was mixed with metal salts (0.4g, 0.6g, 0.8g) in a mass ratio of 1:(2-4). The mixture was reacted for 30 minutes according to steps 1 and 2). The precipitate was washed by centrifugation and dried to constant weight. The mass of the dried tannic acid metal complex was weighed and the reaction yield was calculated.

[0059] 2) Test Results

[0060] The reaction yields of the complexes were determined and are shown in Table 1.

[0061] Table 1. Yields of tannic acid metal complexes formed by the reaction of tannic acid with different metal salts.

[0062] Tannic acid: Ferric sulfate = 1:2 0.1978±0.0101 32.97 Tannic acid: Zinc acetate = 1:2 0.2192±0.0074 36.53 Tannic acid:copper sulfate = 1:2 0.0016±0.0010 0.27 Tannic acid: Ferric sulfate = 1:3 0.0570±0.0053 7.13 Tannic acid: Zinc acetate = 1:3 0.2972±0.0061 37.15 Tannic acid:copper sulfate = 1:3 0.0733±0.0043 9.16 Tannic acid: Ferric sulfate = 1:4 0.0218±0.0021 2.18 Tannic acid: Zinc acetate = 1:4 0.3583±0.0052 35.83 Tannic acid:copper sulfate = 1:4 0.1024±0.0065 10.24

[0063] Ferric sulfate, copper sulfate, and zinc acetate all reacted with tannic acid to produce precipitates, which were black, dark green, and white, respectively, indicating that tannic acid formed insoluble complexes with the metal ions. Among these, the average yields of the complexes were 0.27%–10.24% and 2.18%–32.97% when copper sulfate and ferric sulfate were used as crosslinking agents, respectively, at different ratios of tannic acid to metal salt. The reaction of zinc acetate with tannic acid yielded the highest amount of zinc tannate complex, with corresponding yields of 35.83%–37.15%.

[0064] III. The Influence of Surfactant Type on the Morphology of Functionalized Nanoparticles Carrying Metals

[0065] 1) Experimental Results

[0066] Surfactants OP-10#, 4917#, OP-21#, 1086#, 602#, and 3300# were prepared into 200 mL solutions with concentrations of 0.1%, 1%, and 6%, respectively. Nanoparticles carrying the drug were added at 0.1% of each solution and ultrasonically dispersed at room temperature. Then, 10 mL of a 2% tannic acid aqueous solution was added, followed by dropwise addition of 10 mL of a 6% zinc acetate aqueous solution. After the reaction, the mixture was centrifuged and dried to obtain functionalized nanoparticles carrying the drug.

[0067] 2) Test Results

[0068] The electron microscopy results are as follows:

[0069] Scanning electron microscopy revealed that different mass fractions (0.1%, 1%, 6%) of surfactant OP-10# were used. Figure 2 A), 4917# Figure 2 B), 1086# Figure 2 D), 3300# Figure 2 F) Dispersing the nano-metal drug-loaded particles revealed varying degrees of leakage of the octahedral nano-metal drug-loaded particles, which were not completely encapsulated by the zinc tannate shell. Additionally, the surfactant OP-21# caused the zinc tannate to spherically form individual particles. Figure 2 C) indicates that different types of surfactants have significantly different dispersing properties. Through screening different types of surfactants, surfactant 602# was found to be the most effective. Figure 2 E) The zinc tannate shell can encapsulate most of the nano-sized metal drug-loaded particles, and the particle size distribution is uniform. Furthermore, the appearance morphology of the nano-sized metal drug-loaded particles encapsulated with surfactant 602# at three different mass fractions of 0.1%, 1%, and 6% showed no significant difference.

[0070] IV. Effects of Surfactant Dosage and Ultrasonic Time on Drug Loading of Functionalized Nanoparticles

[0071] 1) Test methods

[0072] Using tebuconazole as the active ingredient, nano-metal drug-loaded particles loaded with tebuconazole were prepared.

[0073] Metal particles loaded with pesticides were added to solutions of surfactant 602# (0.1%, 1%, 6%) with different mass fractions and reacted at room temperature. A series of ultrasonic times were set: 10 min, 30 min, 60 min, and 120 min to prepare functionalized nano-metal drug-loaded particles, and the drug loading was detected.

[0074] The method for determining the pesticide loading is as follows: Weigh 0.04 g (accurate to 0.0005 g) of each of the following pesticide-loaded particles: styraxazole nano-metallic particles, styraxazole functionalized nano-metallic particles, and fipronil functionalized nano-metallic particles, into 25 mL brown volumetric flasks. Add an appropriate amount of methanol to dissolve the pesticide using ultrasonication. Cool to room temperature, dilute to the mark with methanol, filter through a 0.45 μm filter membrane, and then perform high-performance liquid chromatography (HPLC) detection. Specific chromatographic conditions are as follows:

[0075] Mobile phase: methanol: pure water = 80:20 (volume ratio); flow rate: 1 mL / min; injection volume: 5.0 μL; column temperature: room temperature (temperature variation should not exceed 2℃); detection wavelength: 222 nm (tebuconazole) and 270 nm (dinotefuran); retention time of tebuconazole is approximately 9.7 and 10.7 min, and retention time of dinotefuran is approximately 2.7 min.

[0076] 2) Test Results

[0077] Table 2. Effects of surfactant dosage and ultrasonic time on drug loading (using tebuconazole as an example)

[0078]

[0079]

[0080] Taking tebuconazole as an example, the pesticide loading was optimized using two key factors: the surfactant ratio and the ultrasonic time. Table 2 shows that when the amount of surfactant 602# is constant, the pesticide loading gradually increases to a peak within 10-60 minutes with increasing ultrasonic time. However, after continuing ultrasonication for 120 minutes, the pesticide loading decreases. This is because the surfactant, along with the increasing ultrasonic time, effectively disperses the aggregated nano-metal pesticide particles in the solution, providing favorable conditions for the zinc tannate shell to encapsulate the nano-metal pesticide particles. However, an ultrasonic time of 120 minutes leads to leakage of the active ingredient loaded in the nano-metal framework into the solution, resulting in a decrease in pesticide loading. Therefore, under the experimental conditions, the highest pesticide loading was achieved with an ultrasonic time of 60 minutes.

[0081] After ultrasonic treatment for the same period, the metal-loaded particles were dispersed in surfactant solutions of different concentrations and assembled into a tannic acid metal shell. The pesticide loading was tested, and the highest loading was achieved with 1% surfactant. The reason for this is that sufficient dispersion of the pesticide-loaded particles can be obtained under this condition. However, with 0.1% surfactant, there is still insufficient dispersion of the nano-metal framework carrier. When the surfactant concentration is too high, such as 6%, it may accelerate the leakage of the active pesticide components from the nano-metal framework carrier into the solution.

[0082] In summary, the optimal preparation conditions for functionalized metal nanoparticles are as follows: 200 mg of metal nanoparticles (loaded with tebuconazole or fipronil) are added to a 200 mL solution containing 1% (w / w) surfactant 602#, and sonicated at room temperature for 60 min; 10 mL of 2% tannic acid aqueous solution is added, followed by 10 mL of 6% zinc acetate aqueous solution dropwise. After stirring at 1200 rpm for 5 min, the mixture is vortexed for 30 min, centrifuged at 8000 rpm to obtain a precipitate, washed three times, and dried in an oven at 60℃ for 5 h to obtain functionalized metal nanoparticles.

[0083] Based on the above-mentioned optimal preparation process, tebuconazole nanoparticles, tebuconazole-functionalized nanoparticles, and dinotefuran-functionalized nanoparticles were prepared. The drug loading was determined by high-performance liquid chromatography (HPLC). The optimal loading of tebuconazole in the tebuconazole nanoparticles was 14.32%, the optimal loading of tebuconazole in the tebuconazole-functionalized nanoparticles was 10.55%, and the optimal loading of dinotefuran in the dinotefuran-functionalized nanoparticles was 16.74%.

[0084] Example 3: Characterization of Functionalized Nanomaterial Drug-Loaded Particles

[0085] I. Surface observation of nano-metal drug-loaded particles using transmission electron microscopy and scanning electron microscopy

[0086] Example 2: The functionalized nanomaterials prepared under optimal conditions have a spherical structure. Figure 3 A, B), median diameter D 50 142.7nm ( Figure 3 C).

[0087] II. Structural Characterization of Functionalized Nanomaterial Drug-Loaded Particles

[0088] Using Fourier transform infrared spectroscopy (FTIR) Figure 4 A) X-ray photoelectron spectroscopy ( Figure 4 B) and EDS energy spectrum ( Figure 4 DF analysis was used to characterize the functionalized nanomaterial-based drug-loaded particles. Results showed that tebuconazole was successfully loaded onto the nanomaterial-based metal framework, and TA-Zn...II Successfully modified onto nano-metal drug-carrying particles.

[0089] Thermogravimetric analysis was used to analyze the results ( Figure 4 C) shows that, compared with the technical grade of tebuconazole, the decomposition temperature of tebuconazole in functionalized nano-metallic drug-loaded particles is significantly increased. This phenomenon is attributed to TA-Zn. II The protective function of the outer shell.

[0090] Example 4: Verification of the environmental response characteristics of the nano-metal drug-loaded particles of the present invention

[0091] I. Dynamic dialysis method for determining the in vitro release of nano-metal drug-loaded particles

[0092] Response temperature and pH release characteristics of nano-metal drug-loaded particles and functionalized nano-metal drug-loaded particles: 30% ethanol aqueous solutions at different temperatures (10℃, 20℃, and 30℃) and pH values ​​(pH=5, pH=7, and pH=9) were prepared as release media. 100 mg of nano-metal drug-loaded particles (with a drug loading rate of 14.32% obtained under optimal conditions in Example 2) and functionalized nano-metal drug-loaded particles (with a drug loading rate of 10.55% obtained under optimal conditions in Example 2) were weighed and loaded into dialysis membranes (8000-14000 KDA), placed in beakers containing 200 mL of release media, and stirred at 100 rpm at room temperature. At regular intervals, 0.5 mL of release media was taken for high-performance liquid chromatography (HPLC) analysis, and the same volume of media was added back in. The cumulative release rate formula is:

[0093]

[0094] Where E r This refers to the cumulative release (%) of a certain type of azole, V e The volume (V) of the release medium collected at certain time intervals e =0.5mL), V0 is the total volume of the release medium (200mL); C n (mg / mL) is the concentration of the tebuconazole in the release medium at time n; m pesticide (mg) is the total mass of mycotoxin in the nano-pesticide.

[0095] II. Test Results

[0096] Under different temperature conditions, the cumulative release efficiency of functionalized nanomaterials carrying drugs ranged from 5.8% to 47.9% after 72 hours, while that of unassembled TA-Zn was lower. H The nano-metallic drug-loaded particles in the outer shell rapidly release the cyproconazole, with a cumulative release efficiency as high as 8.0%-70.6%. The higher the temperature, the faster the release.

[0097] The longer the release time, the more significant the difference. Figure 5 (A, B). Under different pH conditions, the cumulative release rate of tebuconazole from the nano-metal drug-loaded particles was 38.8%-76.2% after 170 h, while the release efficiency of the functionalized nano-metal drug-loaded particles was only 31.7%-45.7%. Figure 5 D, E).

[0098] After 120 hours, the release rate of functionalized nano-metal drug-loaded particles at 30℃ increased by 25.9% and 43.5% compared to those at 20℃ and 10℃, respectively. The final cumulative release rate at pH 5 was 59.0%, while the release efficiencies at pH 7 and 9 were only 45.9% and 37.9%, respectively. The release curves at various temperatures and pH values ​​best conformed to the Peppas kinetic equation. Figure 5 C, F). This proves the addition of TA-Zn. II The outer shell can prolong the release time of the cyproconazole, and the functionalized nano-metal drug-loaded particles have temperature and pH responsive properties.

[0099] Increasing temperature and decreasing pH value are beneficial for the release of pesticide active ingredients from the pesticide-loaded particles.

[0100] Example 5: Verification of the enhanced antibacterial effect of the functionalized nano-metal drug-loaded particles of the present invention under acidic conditions.

[0101] I. Determination of Antibacterial Activity of Functionalized Metal Nanoparticles by Mycelial Growth Rate Method

[0102] PDA media containing tebuconazole technical, tebuconazole suspension, and functionalized metal nanoparticles were prepared to contain active ingredient concentrations of 0.005, 0.01, 0.05, 0.1, and 0.5 μg / mL, respectively. PDA media containing unloaded functionalized metal nanoparticles were prepared to contain concentrations of 0.01, 0.05, 0.1, 0.5, and 1 μg / mL as controls. The pathogen of rice bakanae disease, *Fusarium fujikura*, strain HLB-19, was inoculated. Each treatment was repeated three times. After 6 days of culture, the colony diameter was measured using the cross-cross method, and the EC50 of different treatments was calculated. 50 .

[0103] II. Effects of different pH values ​​on the antibacterial activity of functionalized nanoparticles loaded with metals

[0104] The pH values ​​of PDA medium were adjusted to 5, 7, and 9 using hydrochloric acid and sodium hydroxide, respectively. The drug treatment was set up according to step one, and pathogens were inoculated. The effects of different pH conditions on the antibacterial activity of functionalized nano-metal drug-loaded particles were measured.

[0105] III. Effect of pH on sporulation of Fusarium oxysporum

[0106] The pH values ​​of mung bean liquid culture medium were adjusted to 5, 7, and 9 using hydrochloric acid and sodium hydroxide, respectively, and then sterilized by moist heat. Pre-cultured *Fusarium oxysporum* fungi were punched into mycelial cakes using a 5mm diameter punch and then added to liquid culture media at different pH values, shaken (25℃, 120 rpm / min), with 5 replicates. Sporulation was measured after 5 days. The effect of different pH values ​​on the sporulation yield of *Fusarium oxysporum* was compared.

[0107] IV. Test Results

[0108] 1) Antibacterial activity of functionalized metal nanoparticles and the effect of pH value

[0109] The effects of tebuconazole technical, tebuconazole suspension, and functionalized nano-metal drug-loaded particles (pH=7) on E. elegans of Fusarium oxysporum. C50 The values ​​are 0.033, 0.062, and 0.042, respectively.

[0110] like Figure 6 As shown, since the release of the active ingredient from the functionalized metal nanoparticles may take some time, the antibacterial activity of the functionalized metal nanoparticles and the tebuconazole suspension is slightly lower than that of the tebuconazole technical at the same concentration.

[0111] like Figure 7 As shown, functionalized empty metal nanoparticles (functionalized empty metal nanoparticles prepared according to the optimal conditions of Example 2, wherein no pesticide active ingredient was added) also exhibited certain antibacterial activity against Fusarium oxysporum. This may be due to the Zr in the metal framework carrier and the shell. 4+ and Zn 2+ Ions possess antibacterial activity, thus synergistically enhancing the effects of bactericides. This result can explain why functionalized nanoparticles carrying drug have higher bioactivity than tebuconazole suspensions.

[0112] 2) Effect of different pH values ​​on sporulation of Fusarium oxysporum

[0113] EC of functionalized metal nanoparticles at pH 5 50 The concentration was 0.038 μg / mL, while the EC at pH=7 and pH=9 was... 50 The concentrations were 0.042 μg / mL and 0.057 μg / mL, respectively, under acidic conditions. 50 The lowest sporulation rate indicates that under these conditions, the release of active ingredients from functionalized nanoparticles carrying drugs can be promoted, thereby improving the bactericidal effect. Furthermore, sporulation tests showed that *Fusarium oxysporum* sporulation in an acidic environment at pH 5 was 3.7 × 10⁻⁶. 6 spores / mL, relatively neutral (2.4 × 10⁻⁶). 6 1.8 × 10⁶ spores / mL) and alkaline (1.8 × 10⁶ spores / mL)6 The conditions for spores / mL are even higher. Example 4 results show that an acidic environment enables functionalized nano-metal drug-loaded particles to rapidly release tebuconazole, effectively controlling the enhanced sporulation capacity of pathogens under acidic conditions, thus playing a role in intelligent disease prevention. In rice seedlings, flooding commonly leads to excessive acid production due to anaerobic respiration, causing pathogens to proliferate and exacerbating the damage caused by bakanae disease. Functionalized nano-metal drug-loaded particles can rapidly release pesticide active ingredients in response to acidic conditions, achieving efficient disease control.

[0114] Example 6: Verification of the greenhouse control efficacy of the functionalized nano-metal drug-loaded particles of the present invention against rice bakanae disease.

[0115] I. Inoculation Method

[0116] In a greenhouse pot experiment to assess the efficacy of tebuconazole, 15 mycelial discs (5 mm in diameter) of *Fusarium oxysporum* strain HLB-19 were collected from the edge of the colony and placed in 300 mL Erlenmeyer flasks containing 100 mL of mung bean soup culture medium. The flasks were incubated at 25°C and 120 rpm for 5 days. The spore suspension was filtered through sterile gauze, and 100 μL of the spore solution was transferred to a hemocytometer to count the number of spores. The spore suspension was then prepared into 10... 6 Units / mL are available for use.

[0117] II. Method of Medication

[0118] The effective dosage range of the registered tebuconazole product in the control of rice bakanae disease is 0.15-0.25 g / 100 kg seeds. Three dosages were selected: low concentration (0.15 g / 100 kg seeds), medium concentration (0.20 g / 100 kg seeds), and high concentration (0.25 g / 100 kg seeds). The efficacy of functionalized nano-metallic drug-loaded particles (functionalized nano-metallic drug-loaded particles with a drug loading rate of 10.55% obtained under optimal conditions in Example 2) and tebuconazole suspension (10% commercially available tebuconazole seed treatment suspension) against bakanae disease in greenhouse conditions was tested. Seeds were surface-sterilized with 75% alcohol for 2-3 minutes and rinsed twice with sterile water. As a control group without any added agents, seeds were soaked in a spore suspension for 24 hours, and then coated with functionalized nano-metallic drug-loaded particles (of this invention) and tebuconazole suspension.

[0119] III. Methods for investigating the efficacy of bakanae disease control

[0120] Rice seeds coated with different concentrations of pesticides and control varieties were directly sown, and the control effect was observed during the seedling stage. The main symptoms of bakanae disease were: excessive vegetative growth, weak plants, thin and pale green leaves, and seedling death. The total number of rice plants was surveyed, the number of diseased plants was recorded, and the incidence rate was calculated.

[0121] IV. Methods for determining zinc ion release concentration

[0122] Weigh 300 mg of functionalized metal nanoparticles (functionalized metal nanoparticles with a drug loading rate of 10.55% obtained under optimal conditions in Example 2) and load them into a dialysis membrane (8000-14000 KDA). Place the membrane into a beaker containing 200 mL of release medium (deionized water: ethanol = 70:30) and stir at 100 rpm at room temperature. At regular intervals, take 5 mL of the release medium for ICP / MS determination of zinc ion concentration (replenish with 5 mL of the original buffer solution immediately after each sampling). Repeat three times. Calculate the cumulative zinc ion release using the following formula:

[0123]

[0124] Where E r It is the cumulative release of zinc ions (%), V e The volume (V) of the release medium collected at certain time intervals e =5mL), V0 is the total volume of the release medium (200mL); C n (mg / mL) is the concentration of zinc ions in the release medium at time n; m Zinc ion (mg) is the total mass of zinc ions in functionalized nano-metal drug-loaded particles.

[0125] V. Results of Functionalized Nanoparticles with Metallic Drug-Loaded Particles in the Prevention and Control of Bakanae Disease

[0126] The functionalized nano-metal-loaded drug-loaded particles showed control efficacy of 84.09%, 90.00%, and 93.10% against bakanae disease at low, medium, and high concentrations (0.25 g / 100 kg seeds), respectively. In contrast, the control efficacy of tebuconazole suspension at the same dosage was 81.82%, 84.00%, and 84.48%, respectively, indicating that the functionalized nano-metal-loaded drug-loaded particles have better disease control effects (Table 3).

[0127] Table 3. Comparison of the efficacy of tebuconazole-functionalized nano-metallic drug-loaded particles and tebuconazole suspension against bakanae disease.

[0128]

[0129] The concentration of zinc ions in the solution under different pH and temperature conditions was determined using ICP / MS. The results showed that the carrier system gradually releases zinc ions into the solution (Tables 4 and 5). Increasing temperature and decreasing pH increased the amount of zinc ions released. Zinc is well known to have antifungal properties and can combat a variety of fungi. Therefore, the zinc ions in the shell can synergistically enhance the bactericidal effect of the active ingredient.

[0130] Table 4. Release of zinc ions from functionalized nanoparticles carrying drugs under different pH conditions.

[0131] 0.5 5 5.09 2 5 20.80 3 5 20.84 4 5 24.54 5 5 26.06 6 5 26.89 0.5 7 0.35 2 7 0.65 3 7 0.47 4 7 1.39 5 7 1.59 6 7 1.70 0.5 9 0.14 2 9 0.18 3 9 0.18 4 9 0.26 5 9 1.53 6 9 1.47

[0132] Table 5. Release of zinc ions from functionalized nanomaterial drug-loaded particles under different temperature conditions.

[0133] 0.5 10 0.96 3 10 1.78 4 10 2.84 5 10 3.06 0.5 20 1.47 3 20 3.58 4 20 4.64 5 20 4.73 0.5 30 1.79 3 30 3.74 4 30 4.87 5 30 6.02

[0134] Example 7: Verification of the control efficacy of the functionalized nano-metal drug-loaded particles of the present invention against rice planthoppers.

[0135] I. Source of rice planthoppers tested

[0136] In the efficacy test of fipronil, the indoor efficacy of fipronil-functionalized nano-metal loaded particles was evaluated by using naturally occurring rice planthoppers under greenhouse conditions as the control target.

[0137] II. Method of Medication

[0138] The effective dosage range of the registered product of fipronil in the control of rice planthoppers is 800-1100 g / 100 kg of seeds. A dosage of 950 g / 100 kg of seeds was selected for greenhouse efficacy testing of functionalized nano-metal-loaded particles (fipronil nano-metal-loaded particles with a loading rate of 16.74% obtained under optimal conditions in Example 2) and fipronil seed treatment dispersible powder (22% commercially available fipronil seed treatment dispersible powder) against rice planthoppers. Seeds were surface-disinfected with 75% alcohol for 2-3 minutes and rinsed twice with sterile water. A control group without any pesticides was used as a reference. Rice seeds were coated with functionalized nano-metal-loaded particles (of this invention) and fipronil seed treatment dispersible powder.

[0139] III. Survey Methods

[0140] Rice seeds coated with different pesticides and those used as controls were directly sown, and their control effects were observed during the seedling stage. The number of live planthoppers (adults) on the leaves of seeds treated with control (CK) at which a certain population size was formed was used as the baseline population. Surveys were conducted three times, at 14, 28, and 35 days after sowing. The number of live planthoppers on both the upper and lower surfaces of the leaves was recorded in detail.

[0141] IV. Test Results

[0142] The control efficacy of fipronil suspension concentrate ranged from 72.20% to 79.10%, while that of functionalized nano-metallic pesticide-loaded particles ranged from 68.60% to 83.36%. At 14 days, the control efficacy of fipronil functionalized nano-metallic pesticide-loaded particles was slightly lower than that of the suspension concentrate treatment, which is related to the slow-release properties of nano-pesticides. However, the sustained efficacy was significantly better than that of the suspension concentrate treatment, as evidenced by the superior control effect against rice planthoppers observed at 28 and 35 days (Table 6).

[0143] Table 6. Comparison of the control efficacy of fipronil functionalized nano-metallic drug-loaded particles and fipronil suspension against rice planthoppers.

[0144]

[0145] Example 8: Safety evaluation of the functionalized nano-metal drug-loaded particles of the present invention on rice seedlings

[0146] I. Experimental Methods

[0147] The effective dosage range of the registered tebuconazole product for the control of rice bakanae disease is 0.15-0.25 g / 100 kg seeds. The highest dose was used as the minimum concentration (0.25 g / 100 kg seeds). Safety tests were conducted on the seed coating of functionalized nano-metal-loaded drug particles and tebuconazole suspension at 2 times (0.50 g / 100 kg seeds) and 4 times (1 g / 100 kg seeds). Water treatment served as a control. Paper bed sowing was used, with 30 seeds sown per dish, replicated 4 times. Germination rate was measured at 4-5 days. Moist direct sowing was used, with 10 seeds sown per pot in paddy soil, replicated 4 times. Emergence rate was recorded at the 2-3 leaf stage or 21 days after sowing. Plant height, root length, and fresh weight of rice seedlings were measured at 7, 14, 21, and 35 days.

[0148] II. Test Results

[0149] As shown in Table 7, compared with the control (CK) (95.3%), the germination rate of functionalized nano-metal drug-loaded particles was 95.3%-96.0%, indicating that the functionalized nano-metal drug-loaded particles did not have an adverse effect on seed germination. The germination rate of seeds treated with tebuconazole suspension ranged from 89.3% to 92.7%, a decrease of 2.6%-6.0%. Figure 8 A).

[0150] The seed germination rate of seeds treated with functionalized nano-metal drug-loaded particles was 88.0%-92.7%, the seed germination rate of seeds treated with tebuconazole suspension was 76.0%-84.7%, and the germination rate of the control was 86.7% (Table 8). Figure 8 B). The results showed that treatment with functionalized nano-metallic drug-loaded particles improved the emergence rate of rice seeds, while the suspension agent exhibited an inhibitory effect. Root length, plant height, and fresh weight of rice seedlings were measured at 7, 14, 21, and 35 days after sowing. Figure 9 ).

[0151] Compared with the control, rice seedlings treated with tebuconazole suspension showed lower physiological indicators such as plant height, root length, and fresh weight, indicating that tebuconazole suspension has an inhibitory effect on rice growth, while functionalized nano-metallic drug-loaded particles have a promoting effect on plant growth. This may be because the functionalized nano-metallic drug-loaded particles have a better dispersion and sustained-release effect on tebuconazole, reducing the impact of local high-concentration doses of the agent on the seeds, thereby improving the safety of the agent for seedlings.

[0152] It is well known that polyphenols inhibit plant growth. However, experiments have shown that the metal polyphenol complex of this invention, acting as the outer shell of functionalized nano-metal drug-carrying particles, can significantly promote growth. Furthermore, zinc is an essential trace element for normal plant development and growth; therefore, in practical applications, due to the uniform dispersion and protection of the active ingredient by the nanoparticles, it offers better safety for rice seed germination, seedling emergence, and plant height, and is generally superior to tebuconazole suspension.

[0153] Table 7. Effects of Functionalized Nanomaterial Drug-Loaded Particles on Rice Seed Germination Rate

[0154]

[0155] Table 8. Effects of functionalized nanomaterials carrying drugs on rice seed emergence rate

[0156]

Claims

1. A method for preparing functionalized nano-metal drug-loaded particles, characterized in that, Includes the following steps: 1) Dissolve the active ingredient of pesticide in N,N-dimethylformamide, then add a nano-metal framework carrier, sonicate and stir at room temperature to adsorb, and obtain nano-metal drug-loaded particles. 2) The nano-metal drug-carrying particles obtained in step 1) are modified with tannic acid and metal salt as modifiers to prepare functionalized nano-metal drug-carrying particles; wherein the mass ratio of the pesticide-loading nano-metal framework carrier, tannic acid and metal salt is 1:1:(2~4). The method for modification using tannic acid and metal salts as modifiers in step 2) includes the following steps: A) A nano-metal framework carrier loaded with pesticide and a surfactant solution are mixed at a mass ratio of 1:1000 and ultrasonically dispersed at room temperature to prepare a dispersion of nano-metal pesticide-loaded particles; the ultrasonic time is 60 min, and the mass percentage concentration of the surfactant solution is 1%; the surfactant is 602#. B) Add tannic acid aqueous solution to the nano-metal drug-loaded particle dispersion obtained in step A), and add metal salt aqueous solution dropwise. After stirring at 1200 rpm for 5 min, vortex for 30 min, centrifuge to obtain precipitate, and wash three times with ethanol to remove unreacted tannic acid and metal salt to obtain functionalized nano-metal drug-loaded particles; the metal salt is zinc acetate. The preparation method of the nano-metal framework carrier includes the following steps: a) Dissolve zirconium tetrachloride in N,N-dimethylformamide, and then add terephthalic acid to obtain a mixture; b) Add glacial acetic acid to the mixture obtained in step 1) and carry out a hydrothermal reaction to prepare a nano-metal framework carrier; In steps a) and b), the mass ratio of zirconium tetrachloride, terephthalic acid and glacial acetic acid is (1~2):1:(20~40).

2. The method according to claim 1, characterized in that, In step 2), the mass ratio of the pesticide-loaded nano-metal framework carrier, tannic acid, and metal salt is 1:1:

3.

3. Functionalized nanomaterial drug-loaded particles prepared by the method of claim 1 or 2.

4. The application of the method according to claim 1 or 2 or the functionalized nano-metal drug-loaded particles according to claim 3 in the preparation of nano-pesticides.

5. The application of the method of claim 1 or 2 or the functionalized nano-metal drug-loaded particles prepared therefrom in seed treatment; the seed treatment method includes, but is not limited to, seed soaking, seed coating, and seed pelleting; the purpose of the seed treatment is to promote the germination, emergence and growth of rice seeds, as well as to prevent and control rice diseases and pests.

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