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

Abstract

The invention 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 control of rice diseases and insect pests. According to the invention, the nano-carrier is prepared from the organic metal framework and is loaded with the pesticide, and the tannic acid metal complex shell is assembled, so that targeted controlled release of the pesticide can be carried out at harmful organism hazard parts, and mass propagation of pathogenic bacteria under an acidic condition and harm of pests are effectively coped with. The nano-metal drug-loaded particles show an excellent targeted pest control effect, the effective utilization rate of pesticide active ingredients is increased, the lasting period is prolonged, germination of rice seeds and growth of rice seedlings are promoted, and the nano-metal drug-loaded particles are particularly suitable for improving the quality of the rice seedlings in direct seeding cultivation of the rice and have a wide rice seed treatment application prospect.

Description

Technical Field

[0001] The present invention relates to the field of pesticide preparation processing and application, and specifically to the preparation of rice seed treatment nanopesticides, the prevention and control of rice seedling pests and diseases, and the improvement of rice seedling quality. The present invention can provide a nanopesticide treatment technology for rice seeds, which is suitable for direct seeding and efficient prevention and control of seedling pests and diseases, and is conducive to the simplification of rice cultivation. Background Art

[0002] Rice is an important staple food crop in my country, and the health of its seedlings is crucial to rice yield. The rice seedling stage is affected by a variety of harmful organisms such as seed-borne and soil-borne diseases and pests, and is a critical period for pest and disease control. In particular, the continuous development of rice seed direct seeding and drone seeding technology in recent years has provided an important opportunity for simplified cultivation. Direct-seeded rice often has problems such as weak buds, uneven emergence, shallow roots, and easy lodging of plants. There is an urgent need for the development of related supporting seed treatment technologies and products. Using pesticides to treat seeds can effectively reduce the damage caused by pests and diseases in the seedling stage of crops. Through seed coating, seed mixing, seed soaking, seed pelleting and other seed treatments, concealed pesticide application can be achieved, saving effort and labor. However, the current domestic seed coating agents are mainly suspension agents, with a single dosage form, and the effective ingredients have poor inhibitory effects on pathogens hidden inside the seeds, resulting in serious drug resistance. In addition, drug damage caused by seed soaking also occurs from time to time. Therefore, the development of efficient and safe pesticide formulations that can promote seedling growth is the key to solving the above problems.

[0003] Nanomaterials have small particle size, large specific surface area, can be loaded with pesticides, have strong targeted transport and conduction capabilities for harmful organisms, can release the active ingredients of pesticides on demand, or release them in response to various stimuli (pH value, temperature, humidity, light, etc.) at a specific time and space, and are an effective way to achieve efficient control of harmful organisms by pesticides, and are safe for the environment and non-target organisms. In addition, some nanomaterials have the effects of sterilization and disease prevention, promoting plant growth, and increasing crop biomass and fruit quality. However, there are few reports on the application of nanopesticides in seed treatment, especially nanocarriers and preparations that have the functions of controlling harmful organisms, achieving seed priming, and enhancing seed vitality. Summary of the invention

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

[0005] The object of the present invention is achieved in that:

[0006] In a first aspect of the present invention, a nano-metal skeleton is provided as a carrier, and a method for preparing the nano-metal skeleton carrier comprises the following steps:

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

[0008] 2) adding glacial acetic acid to the mixture obtained in step 1) for hydrothermal reaction to prepare a nano metal skeleton carrier;

[0009] In the method, in step 1) and step 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 the step 1), zirconium tetrachloride (35 mg), terephthalic acid (25 mg) and N,N-dimethylformamide (10 mL) are mixed and dissolved by ultrasonication;

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

[0012] Furthermore, in a second aspect of the present invention, functionalized nano-metal drug-carrying particles are provided, wherein the functionalized nano-metal drug-carrying particles are prepared by the following method:

[0013] 1) dissolving the active ingredient of the pesticide in N,N-dimethylformamide, adding the nano metal skeleton carrier, stirring and adsorbing at room temperature after ultrasonication, and obtaining 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 the step 2), the mass ratio of the nano metal drug-carrying particles, tannic acid, and metal salt is 1:1:(2-4).

[0016] In the above step 2), the method of modifying with tannic acid and metal salt as the modifying substances comprises the following steps:

[0017] A) mixing the nano-metal skeleton carrier loaded with pesticide and the surfactant solution in a mass ratio of 1:1000, and ultrasonically dispersing them at room temperature to prepare a nano-metal drug-loaded particle dispersion;

[0018] B) adding an aqueous solution of tannic acid to the dispersion of the nano-metal drug-loaded particles obtained in step A), and then adding an aqueous solution of a metal salt dropwise, stirring at 1200 rpm for 5 minutes, vortexing for 30 minutes, centrifuging to obtain a precipitate, and washing three times with ethanol to remove unreacted tannic acid and metal salt, thereby obtaining functionalized nano-metal drug-loaded particles;

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

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

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

[0022] In one embodiment of the present invention, the nano-metal drug-carrying particles have ipconazole or dinotefuran as the active ingredient, and the mass ratio of the pesticide active ingredient to the nano-metal skeleton carrier is 1:2.

[0023] The advantages of the present invention are:

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

[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 amount and are more in line with the needs of industrial production.

[0026] 3) The drug loading of the functionalized nanometal drug-loaded particles obtained by the above loading method is 10.55±0.48%. Compared with the original metal organic framework (MOFs) method, the open embedding of pesticides is transformed into a closed package, which significantly improves the controlled release effect of pesticides in response to pH and temperature. On the one hand, this increases the drug loading capacity of the metal skeleton, avoids the waste of carriers and the increase in costs in production, and makes the application of this nanocarrier in the field of pesticides possible. In addition, it also achieves effective control of pathogens that are controlled in large quantities under acidic conditions, and improves seed vitality and seedling quality by triggering the breaking of seed dormancy.

[0027] 4) The median diameter of the functionalized metal-loaded nanoparticles prepared by the above method is 142.7 nm. The nanopesticide can have a good controlled release effect in the response environment, which is conducive to improving the utilization rate of pesticides, precise prevention and control, and extending the effective period, and has broad development and application prospects.

[0028] 5) Metal organic frameworks (MOFs) are porous nanomaterials that achieve structural diversity by selecting different metal ions and organic ligands. They also have inherent advantages such as high porosity, large specific surface area, and stable structure. When nano-metal pesticide particles formed by loading pesticides on nano-metal organic frameworks are used for seed treatment, a layer of pesticide attachment will be formed on the surface of the seeds. Nano-metal organic particles can fully enrich nutrients, continuously provide nutrients during seed germination and growth, and promote the absorption of nutrients. In addition, the structure has an antibacterial effect, preventing pathogenic microorganisms and pests in the soil from harming seeds and seedlings, and playing a role in preventing diseases and resisting insects. Plant seeds treated with nano-metal drug-loaded particles are not easily attacked by pests during storage, which is conducive to long-term storage and transportation, and has important economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Microscopic morphology (AC) of nanometal skeleton carriers prepared with 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 microscope and particle size distribution (D) of nanometal skeleton carriers.

[0030] Figure 2 The microscopic morphology of functionalized nanometal drug-loaded particles under scanning electron microscope was 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%.

[0031] Figure 3 Microscopic morphology of functionalized nano-metal drug-loaded particles under scanning electron microscopy (A) and transmission electron microscopy (B) and particle size distribution of functionalized nano-metal drug-loaded particles (C).

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

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

[0034] Figure 6 The inhibitory effects of ioconazole original drug, ioconazole suspension and functionalized nano-metal drug-loaded particles on the in vitro mycelial growth of Fusarium fujikura at different concentrations of ioconazole active ingredients. And the inhibitory effects of functionalized nano-metal drug-loaded particles on the in vitro mycelial growth of Fusarium fujikura under different pH conditions.

[0035] Figure 7 The inhibitory effect of different concentrations of functionalized nanometal empty particles (without added pesticide active ingredients) on the in vitro mycelial growth of Fusarium fujikura.

[0036] Figure 8 Effects of iodinaprole on the safety of rice germination (A) and emergence (B). a, b, c are low, medium, and high doses of iodinaprole suspension, d, e, f are low, medium, and high doses of functionalized nanometal drug-loaded particles, and g is a blank control.

[0037] Fig. 9 Effects of iobactam suspension concentrate and functionalized metal nanoparticles on rice root length (A), leaf length (B) and fresh weight (C) at low, medium and high doses. DETAILED DESCRIPTION

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

[0039] In the following embodiments, the “parts by mass” mentioned herein is a unit of weight as understood in the art, and may be “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 Xiens Biochemical Technology Co., Ltd.), glacial acetic acid (99%, Tianjin Xiens Biochemical Technology Co., Ltd.), tannic acid (96%, Tianjin Xiens Biochemical Technology Co., Ltd.), zinc acetate (98%, Shanghai McLean Biochemical Technology Co., Ltd.), copper sulfate (99%, Shanghai McLean Biochemical Technology Co., Ltd.), ferric sulfate (97%, Tianjin Xiens Biochemical Technology Co., Ltd.), cyproconazole technical (98%, Lanxess, Germany), dinotefuran (99%, Shandong United Pesticide Industry Co., Ltd.), cyproconazole 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 seeds are Longgeng 31, a conventional japonica rice.

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

[0044] The following uses ipoxadone and dinotefuran as examples to illustrate the preparation method and optimal conditions of the functionalized nano-metal drug-carrying particles of the present invention. It should be known to those skilled in the art that the pesticides that can be loaded into the functionalized nano-metal drug-carrying particles of the present invention include but are not limited to ipoxadone and dinotefuran.

[0045] Example 1. Preparation of Nanometal Skeleton Carrier

[0046] 1. The preparation method of the nano metal skeleton 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 ultrasound;

[0048] 2) Add glacial acetic acid (500 mg, 750 mg, 1000 mg) to the mixture in step 1), that is, the mass ratios of zirconium tetrachloride, terephthalic acid and glacial acetic acid are 1:1:20, 1.4:1:30 and 2:1:40 respectively. The mixture is subjected to hydrothermal reaction in a 120°C oven for 24 hours to synthesize a metal skeleton, centrifuged at 8000 rpm to obtain a precipitate, washed three times with N, N-dimethylformamide to remove the remaining reactants, and placed in a 60°C oven for drying to prepare nano-metal skeleton carrier particles.

[0049] 2. Electron Microscope Observation Method

[0050] The nano-metal skeleton carrier particles were fixed on the sample holder with conductive glue, and after being sprayed with gold, they were placed on the scanning electron microscope sample holder and vacuumed for observation. The test pressure was 2kV.

[0051] 3. Scanning electron microscope observation results: The white powder obtained by steps 1) and 2) has a regular octahedral structure ( Figure 1 AC), the mass ratio of zirconium tetrachloride, terephthalic acid and glacial acetic acid (1:1:20, 1.4:1:30 and 2:1:40) has a slight effect on the morphology of the nano-metal skeleton carrier. According to the morphological characteristics of the nano-metal skeleton carrier, the mass ratio of zirconium tetrachloride, terephthalic acid and glacial acetic acid is preferably 1.4:1:30 to prepare nano-metal skeleton carrier particles for subsequent experiments. The median diameter D 50 84.7nm( Figure 1 D).

[0052] Example 2: Preparation of functionalized metal nanoparticles and optimization of their preparation conditions

[0053] 1. The functionalized nano-metal drug-carrying particles are prepared by using the nano-metal skeleton carrier obtained in Example 1, comprising the following steps:

[0054] 1) Pesticide loading: 2.4 g of ipiconazole or dinotefuran was dissolved in N,N-dimethylformamide (15 mL), and the nano-metal skeleton carrier (4.8 g) prepared in Example 1 was added and stirred for adsorption at room temperature. After loading, the precipitate was centrifuged and washed, and dried in an oven at 60° C. for 3 h to obtain nano-metal drug-loaded particles loaded with pesticides;

[0055] 2) Preparation of functionalized nano-metal drug-loaded particles: A) Mixing the nano-metal drug-loaded particles loaded with pesticides and the surfactant solution in a mass ratio of 1:1000, and fully dispersing them by ultrasound at room temperature to prepare a nano-metal drug-loaded particle dispersion; B) Adding a tannic acid aqueous solution to the nano-metal drug-loaded particle dispersion obtained in step A), and then adding a metal salt aqueous solution dropwise, stirring and reacting at 1200 rpm for 5 minutes, vortexing and oscillating for 30 minutes, and gradually depositing a tannic acid metal complex shell on the surface of the metal drug-loaded particles. After centrifugation at 8000 rpm, the precipitate was collected, washed three times with ethanol to remove unreacted tannic acid and metal salt, and dried in an oven at 60°C for 5 hours to obtain functionalized nano-metal drug-loaded particles.

[0056] 2. Effect of metal salt types on the reaction yield of tannic acid metal complexes

[0057] 1) Test methods

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

[0059] 2) Test results

[0060] After testing, the reaction yield of the complex is shown in Table 1:

[0061] Table 1 The yield of tannic acid metal complexes generated by the reaction of tannic acid with different metal salts

[0062] Tannic acid to metal salt ratio Complex yield (g) Reaction yield (%) Tannic acid: ferrous 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: ferrous 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: ferrous 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] Ferrous sulfate, copper sulfate and zinc acetate reacted with tannic acid to form precipitates, which were black, dark green and white, respectively, indicating that tannic acid formed insoluble complexes with metal ions. Under different ratios of tannic acid to metal salt, the average yields of the complexes when copper sulfate and ferrous sulfate were used as cross-linking agents were 0.27% to 10.24% and 2.18% to 32.97%, respectively. The highest yield of zinc tannate complex was obtained when zinc acetate reacted with tannic acid, and the corresponding yield was 35.83% to 37.15%.

[0064] 3. Effect of surfactant types on the morphology of functionalized metal nanoparticles

[0065] 1) Test 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, and nano-metal drug-carrying particles were added at 0.1%, and ultrasonic dispersion was performed at room temperature. 10 mL of 2% tannic acid aqueous solution was added, and 10 mL of 6% zinc acetate aqueous solution was added dropwise, and functionalized nano-metal drug-carrying particles were obtained after centrifugal drying after the reaction.

[0067] 2) Test results

[0068] The electron microscopy results are as follows:

[0069] Scanning electron microscopy revealed that the surfactant OP-10#( Figure 2 A) 4917#( Figure 2 B) 1086#( Figure 2 D) 3300#( Figure 2 F) When dispersing the nano-metal drug-carrying particles, there are different degrees of nakedness of the regular octahedral nano-metal drug-carrying particles, which are not completely wrapped by the zinc tannate shell. And the phenomenon of zinc tannate forming a single ball caused by surfactant OP-21# ( Figure 2 C), indicating that the dispersing properties of different types of surfactants vary greatly. Through the screening of different types of surfactants, it was found that surfactant 602# had the best effect ( Figure 2 E) The zinc tannate shell can wrap most of the nano-metal drug-loaded particles, and the particle size distribution is uniform. In addition, the surfactant 602# wraps the nano-metal drug-loaded particles at three different mass fractions of 0.1%, 1%, and 6%, and there is no significant difference in appearance.

[0070] 4. Effects of surfactant dosage and ultrasound time on drug loading of functionalized metal nanoparticles

[0071] 1) Test methods

[0072] Taking ipconazole as an effective ingredient, nano metal drug-carrying particles loaded with ipconazole are prepared.

[0073] The 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: 10min, 30min, 60min, 120min to prepare functionalized nano-metal drug-loaded particles and detect the drug loading amount.

[0074] The method for determining the drug loading is as follows: weigh 0.04 g (accurate to 0.0005 g) of ipconazole nano-metal drug-loaded particles, ipconazole functionalized nano-metal drug-loaded particles, and dinotefuran functionalized nano-metal drug-loaded particles respectively into a 25 mL brown volumetric flask, add an appropriate amount of methanol to dissolve the pesticides by ultrasonication, cool to room temperature, dilute to the mark with methanol, and filter through a 0.45 μm filter membrane before high performance liquid chromatography detection. The specific chromatographic detection conditions are:

[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 change should not be greater than 2°C); detection wavelength: 222 nm (iopconazole) and 270 nm (dinotefuran), the retention time of iopconazole is approximately 9.7 and 10.7 min, and the retention time of dinotefuran is approximately 2.7 min.

[0076] 2) Test results

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

[0078]

[0079]

[0080] Taking oxadiazole as an example, the drug loading was optimized by using two key factors, namely the ratio of surfactant and ultrasonic time. It can be seen from Table 2 that when the dosage of surfactant 602# is constant, the drug loading gradually increases and reaches a peak value with the increase of ultrasonic time within 10-60 minutes. When the ultrasonic time is continued to 120 minutes, the drug loading decreases. The reason is that as the ultrasonic time increases and the surfactant acts, the aggregated nano-metal drug-loaded particles are fully dispersed in the solution, providing favorable conditions for the zinc tannate shell to wrap the nano-metal drug-loaded particles. However, when the ultrasonic time is 120 minutes, the active ingredients loaded in the nano-metal skeleton carrier will leak into the solution, and the drug loading will decrease. Therefore, under the experimental conditions, the pesticide loading is the highest when the ultrasonic time is 60 minutes.

[0081] After ultrasonic treatment for the same time, the metal drug-loaded particles were dispersed in surfactant solutions of different concentrations and then assembled into tannic acid metal shells. The loading amount of pesticide was tested, and the highest amount was found when the surfactant dosage was 1%. The reason for this was analyzed to be: under this condition, the drug-loaded particles could be fully dispersed, while at a surfactant dosage of 0.1%, the nano-metal skeleton carrier was still not sufficiently dispersed. When the surfactant concentration was too high, such as at a dosage of 6%, the leakage of the active ingredients of the pesticide in the nano-metal skeleton carrier into the solution might be accelerated.

[0082] In summary, the optimal preparation conditions of functionalized nano-metal drug-loaded particles are as follows: add 200 mg of nano-metal drug-loaded particles (loaded with ioconazole or dinotefuran) to a 1% mass fraction surfactant 602# solution (200 mL), and ultrasonicate at room temperature for 60 min; add 10 mL of 2% tannic acid aqueous solution, and add 10 mL of 6% zinc acetate aqueous solution dropwise, stir at 1200 rpm for 5 min, vortex for 30 min, centrifuge at 8000 rpm to obtain a precipitate, wash three times, and dry in an oven at 60°C for 5 h to obtain functionalized nano-metal drug-loaded particles.

[0083] Based on the above optimal preparation process, cyproconazole nano-metal drug-loaded particles, cyproconazole functionalized nano-metal drug-loaded particles and dinotefuran functionalized nano-metal drug-loaded particles were prepared respectively. The drug loading was detected by high performance liquid chromatography. The optimal loading amount of cyproconazole in cyproconazole nano-metal drug-loaded particles was 14.32%, the optimal loading amount of cyproconazole in cyproconazole functionalized nano-metal drug-loaded particles was 10.55%, and the optimal loading amount of dinotefuran in dinotefuran functionalized nano-metal drug-loaded particles was 16.74%.

[0084] Example 3: Characterization of functionalized metal nanoparticles loaded with drugs

[0085] 1. Surface observation of metal nanoparticles using transmission electron microscopy and scanning electron microscopy

[0086] Example 2 The functionalized nano-metal drug-carrying particles prepared under the optimal conditions are spherical in structure ( Figure 3 A, B), median diameter D 50 142.7nm( Figure 3 C).

[0087] 2. Structural Characterization of Functionalized Metal Nanoparticles

[0088] Using Fourier transform infrared spectroscopy ( Figure 4 A), X-ray photoelectron spectroscopy ( Figure 4 B) and EDS spectrum ( Figure 4 DF) analysis of the characteristics of functionalized nanometal drug-loaded particles. The results showed that ipoxetine was successfully loaded on the nanometal skeleton carrier, and TA-ZnII Successfully modified on nano metal drug-loaded particles.

[0089] Thermogravimetric analysis showed that ( Figure 4 C) shows that the decomposition temperature of iodine in the functionalized nanometal drug-loaded particles is significantly increased compared with that of the original iodine. This phenomenon is attributed to the II The protective function of the shell.

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

[0091] 1. Dynamic dialysis method to test the in vitro release of metal nanoparticles

[0092] Response temperature and pH release characteristics of nanometal drug-loaded particles and functionalized nanometal drug-loaded particles: Prepare 30% ethanol aqueous solution at different temperatures (10°C, 20°C and 30°C) and pH (pH=5, pH=7 and pH=9) as release medium. Weigh 100 mg of nanometal drug-loaded particles (nanometal drug-loaded particles with a drug loading rate of 14.32% obtained under the optimal conditions of Example 2) and functionalized nanometal drug-loaded particles (functionalized nanometal drug-loaded particles with a drug loading rate of 10.55% obtained under the optimal conditions of Example 2) respectively, load them into a dialysis membrane (8000-14000KDA), put them into a beaker containing 200 mL of release medium, and stir at 100 rpm at room temperature. Take out 0.5 mL of release medium at regular intervals for high performance liquid chromatography analysis, and add the same volume of medium. The cumulative release rate formula is:

[0093]

[0094] Where E r is the cumulative release of vinblastine (%), V e is the volume of the release medium collected at certain time intervals (V e =0.5 mL), V0 is the total volume of the release medium (200 mL); C n (mg / mL) is the concentration of ipconazole in the release medium at time n; m pesticide (mg) is the total mass of ioconazole in the nanopesticide.

[0095] 2. Test results

[0096] Under different temperature conditions, the cumulative release efficiency of functionalized metal nanoparticles was as low as 5.8%-47.9% at 72h. H The nano-metal drug-loaded particles in the shell quickly release iodide, with a cumulative release efficiency of up to 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 ipconazole from the nanometal drug-loaded particles was 38.8%-76.2% at 170 h, while the release efficiency of the functionalized nanometal drug-loaded particles was only 31.7%-45.7% ( Figure 5 D, E).

[0098] After 120 hours, the release rate of functionalized metal nanoparticles at 30°C increased by 25.9% and 43.5% compared with that at 20°C and 10°C, respectively. The final cumulative release rate at pH 5 was 59.0%, while the release efficiency at pH 7 and 9 was only 45.9% and 37.9%, respectively. The release curves at each temperature and pH best fit the Peppas kinetic equation ( Figure 5 C, F) proves that adding TA-Zn II The shell can prolong the release time of ipconazole, and the functionalized nanometal drug-loaded particles have temperature and pH responsive properties.

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

[0100] Example 5: Verification of the enhanced antibacterial effect of the functionalized metal nanoparticles under acidic conditions

[0101] 1. Determination of antibacterial activity of functionalized metal nanoparticles by mycelial growth rate method

[0102] The chlorpyrifos original drug, chlorpyrifos suspension and functionalized nanometal drug-loaded particles were respectively formulated into drug-containing PDA medium with active ingredient mass concentrations of 0.005, 0.01, 0.05, 0.1 and 0.5 μg / mL, and the functionalized nanometal empty particles without drug loading were formulated into PDA medium with concentrations of 0.01, 0.05, 0.1, 0.5 and 1 μg / mL as controls. The HLB-19 strain of Fusarium fujikura, the pathogen of rice seedling disease, was inoculated. Each treatment was repeated 3 times. The colony diameter was measured by the cross method after 6 days of culture, and the EC of different drug treatments was calculated 50 .

[0103] 2. Effect of different pH values ​​on the antibacterial activity of functionalized metal nanoparticles

[0104] The pH value of the PDA culture medium was adjusted to 5, 7, and 9 with hydrochloric acid and sodium hydroxide, respectively. The drug treatment was set according to step 1, pathogens were inoculated, and the effects of different pH conditions on the antibacterial activity of functionalized nanometal drug-loaded particles were determined.

[0105] 3. Effect of pH on the spore production of Fusarium fujikura

[0106] The pH value of the mung bean liquid culture medium was adjusted to 5, 7, and 9 with hydrochloric acid and sodium hydroxide, and then sterilized with wet heat for later use. The pre-cultured Fusarium fujikura was punched with a 5 mm diameter puncher, and then placed in liquid culture media with different pH values ​​and shaken (25°C, 120 rpm / min). Five repetitions were set, and the spore production was measured after 5 days. The effects of different pH values ​​on the spore production of Fusarium fujikura were compared.

[0107] 4. Test results

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

[0109] Effects of ipconazole original drug, ipconazole suspension and functionalized nanometal drug-loaded particles (pH = 7) on the E. C50 They are 0.033, 0.062 and 0.042 respectively.

[0110] like Figure 6 As shown in the figure, since it may take some time for the active ingredients to be released from the functionalized nano-metal drug-loaded particles, the antibacterial activity of the functionalized nano-metal drug-loaded particles and ioconazole suspension was slightly lower than that of ioconazole original drug at the same concentration.

[0111] like Figure 7 As shown in the figure, the functionalized nanometal empty particles (the functionalized nanometal empty particles prepared according to the optimal conditions of Example 2, wherein no pesticide active ingredient was added) also showed a certain antibacterial activity against Fusarium fuscata, which may be due to the Zr in the metal skeleton carrier and the shell. 4+ and Zn 2+ The ions have antibacterial activity, thus synergizing the bactericide. This result can be used to explain why the bioactivity of functionalized metal nanoparticles is higher than that of ioconazole suspension.

[0112] 2) Effect of different pH values ​​on the spore production of Fusarium fujikura

[0113] EC of functionalized metal nanoparticles loaded with drugs at pH = 5 50 The EC at pH = 7 and pH = 9 was 0.038 μg / mL. 50 The EC values ​​were 0.042 μg / mL and 0.057 μg / mL respectively. 50 The lowest concentration was 1.7×10-1.5, indicating that under this condition, the release of active ingredients in functionalized nanometal drug-loaded particles can be promoted, thereby improving the bactericidal effect. In addition, the spore production test results showed that the spore production of Fusarium fusariosus in an acidic environment of pH = 5 was 3.7×10 6 spores / mL, relatively neutral (2.4×10 6 spores / mL) and alkaline (1.8×106 Spores / mL) conditions are higher. The results of Example 4 show that the acidic environment enables the functionalized nano-metal drug-loaded particles to quickly release oxazolidinone, which can effectively control the enhanced spore production ability of pathogens under acidic conditions and play a role in intelligent disease prevention. When rice seedlings are flooded, the plants generally produce a large amount of acid due to anaerobic respiration, which leads to the massive reproduction of pathogens and aggravates the harm of seedling blight. Functionalized nano-metal drug-loaded particles can quickly release the active ingredients of pesticides in response to acidic conditions to achieve efficient control of diseases.

[0114] Example 6: Verification of the greenhouse efficacy of the functionalized metal nanoparticles of the present invention against rice bakanae disease

[0115] 1. Inoculation method

[0116] In the greenhouse potted plant protection test of cyproconazole, 15 5mm diameter cakes of Fusarium fujikura HLB-19 strain were taken from the edge of the colony and placed in a 300mL Erlenmeyer flask containing 100mL mung bean soup medium. The spore suspension was filtered through sterile gauze, and 100μL of spore liquid was drawn onto a blood cell counting plate to count the number of spores. The spore suspension was prepared into 10 6 pcs / mL for future use.

[0117] 2. Method of use

[0118] The effective dosage range of the registered product of cyproconazole in the prevention and control of rice seedling disease is 0.15-0.25g / 100kg seeds. Three dosages were selected, namely, low concentration (0.15g / 100kg seeds), medium concentration (0.20g / 100kg seeds) and high concentration (0.25g / 100kg seeds). Functionalized nano-metal drug-loaded particles (functionalized nano-metal drug-loaded particles with a drug loading rate of 10.55% obtained under the optimal conditions of Example 2) and cyproconazole suspension (10% cyproconazole commercial seed treatment suspension) were tested for greenhouse prevention of seedling disease. 75% alcohol was used to disinfect the seeds on the surface for 2-3min and washed twice with sterile water. The control group was used as a control without adding any agent. After the seeds were soaked in the spore suspension for 24h, the rice seeds were coated with functionalized nano-metal drug-loaded particles (the present invention) and cyproconazole suspension.

[0119] 3. Investigation methods for the efficacy of Bakanae disease prevention

[0120] The control and rice seeds coated with different concentrations of different agents were directly planted and their control effects were observed at the seedling stage. The main symptoms of seedling blight are: leggy growth, thin and weak plants, thin and light green leaves, and dead seedlings. All rice plants were surveyed, the number of diseased plants was recorded, and the incidence rate was calculated.

[0121] 4. Determination of zinc ion release concentration

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

[0123]

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

[0125] 5. Results of the efficacy of functionalized metal nanoparticles against Bakanae disease

[0126] The functionalized nano-metal drug-loaded particles had a protective effect against seedling blight at low (0.15 g / 100 kg seeds), medium (0.20 g / 100 kg seeds) and high (0.25 g / 100 kg seeds) concentrations of 84.09%, 90.00% and 93.10% respectively, while at the same dose, the protective effect of iodide suspension was 81.82%, 84.00% and 84.48% respectively, indicating that the functionalized nano-metal drug-loaded particles had a better disease prevention effect (Table 3).

[0127] Table 3 Comparison of the efficacy of oxadiazole functionalized nanometal drug-loaded particles and oxadiazole suspension against seedling bacillus

[0128]

[0129] ICP / MS was used to measure the concentration of zinc ions in the solution under different pH and temperature conditions. The results showed that the carrier system gradually released zinc ions into the solution (Tables 4 and 5). The release of zinc ions can be increased with increasing temperature and decreasing pH. It is well known that zinc has antifungal properties and can fight against a variety of fungi. Therefore, the zinc ions in the shell can synergistically improve the bactericidal effect of the active ingredient.

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

[0131] Time (day) pH Zinc ion release rate (%) 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 metal nanoparticles at different temperatures

[0133] Time (day) Temperature(℃) Zinc release (%) 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 efficacy of the functionalized metal nanoparticles of the present invention in preventing rice planthoppers

[0135] 1. Source of rice planthoppers

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

[0137] 2. Method of use

[0138] The effective dosage range of the registered product of dinotefuran in the control of rice planthoppers is 800-1100g / 100kg seeds. The dosage of 950g / 100kg seeds was selected to test the greenhouse control effect of functionalized nano-metal drug-loaded particles (dinotefuran nano-metal drug-loaded particles with a drug loading rate of 16.74% obtained under the optimal conditions of Example 2) and dinotefuran seed treatment dispersible powder (22% dinotefuran commercial seed treatment dispersible powder) on rice planthoppers. Use 75% alcohol to disinfect the seeds for 2-3 minutes and wash them with sterile water twice. The control group was used as a control without adding any drug, and the rice seeds were coated with functionalized nano-metal drug-loaded particles (of the present invention) and dinotefuran seed treatment dispersible powder.

[0139] III. Survey Methods

[0140] The control and rice seeds coated with different agents were directly planted and their control effects were observed at the seedling stage. The number of live rice planthoppers (adults) on the leaves of CK-treated seeds when a certain population was formed was taken as the insect population base. The survey time was 14, 28, and 35 days after sowing, and a total of 3 surveys were conducted. The number of live rice planthoppers on the front and back of the leaves was recorded in detail.

[0141] 4. Test results

[0142] The control effect of the dinotefuran suspension was 72.20%-79.10%, and the control effect of the functionalized nano-metal drug-loaded particles was 68.60%-83.36%. The control effect of the dinotefuran functionalized nano-metal drug-loaded particles was slightly worse than that of the suspension treatment at 14 days, which is related to the slow release of nanopesticides. The persistence was significantly better than that of the suspension treatment, as shown by the better control effect on rice planthoppers when investigated on the 28th and 35th days (Table 6).

[0143] Table 6 Comparison of the efficacy of dinotefuran functionalized nanometal drug-loaded particles and dinotefuran suspension against rice planthoppers

[0144]

[0145] Example 8: Safety evaluation of the functionalized metal nanoparticles on rice seedlings

[0146] 1. Test methods

[0147] The effective dosage range of registered products of chloramphenicol in the prevention and control of rice seedling disease is 0.15-0.25g / 100kg seeds. The highest dose was used as the minimum drug concentration (0.25g / 100kg seeds), and the seed coating safety test of functionalized nanometal drug-loaded particles and chloramphenicol suspension was carried out at 2 times (0.50g / 100kg seeds) and 4 times (1g / 100kg seeds). The water treatment was used as the control treatment, and the paper bed method was used for sowing. 30 seeds were sown per dish, 4 times were repeated, and the germination rate was measured at 4-5 days. Wet direct seeding was used, sowing was carried out in paddy field soil, 10 seeds were sown per pot, 4 times were repeated, and the emergence rate was counted at 2 to 3 leaf stage or 21 days after sowing. The plant height, root length and fresh weight of rice seedlings were measured at 7 days, 14 days, 21 days and 35 days.

[0148] 2. Test results

[0149] As shown in Table 7, compared with the control CK (95.3%), the germination rate of the functionalized nano-metal drug-loaded particles was 95.3%-96.0%, which indicated that the functionalized nano-metal drug-loaded particles had no adverse effect on seed germination. The germination rate of seeds treated with ioconazole suspension was between 89.3% and 92.7%, and the germination rate decreased by 2.6%-6.0% ( Figure 8 A).

[0150] The germination rates of seeds treated with functionalized nanometal drug-loaded particles were 88.0%-92.7%, those treated with ipconazole suspension were 76.0%-84.7%, and the germination rate of the control was 86.7% (Table 8; Figure 8 B). The results showed that the functionalized nanometal drug-loaded particles increased the germination rate of rice seeds, while the suspension concentrate showed an inhibitory effect. The root length, plant height and fresh weight of rice seedlings were measured at 7, 14, 21 and 35 days after sowing ( Fig. 9 ).

[0151] Compared with the control, the physiological indicators of rice seedlings treated with iodine suspending agent, such as plant height, root length and fresh weight, were lower, indicating that iodine suspending agent had an inhibitory effect on rice growth, while functionalized nano-metal drug-loaded particles promoted plant growth. This may be because functionalized nano-metal drug-loaded particles have a good dispersion and slow release effect on iodine, reducing the local high concentration dose effect of the agent on seeds, thereby improving the safety of the agent on seedlings.

[0152] As is known to all, polyphenols can hinder plant growth. However, experiments have shown that the metal polyphenol complex of the present invention, as the shell of the functionalized nano metal drug-carrying particles, can significantly promote growth. In addition, zinc is a trace element necessary for the normal development and growth of plants. Therefore, in practical applications, due to the uniform dispersion and protection of the active ingredients by the nanoparticles, it has better safety for the germination, emergence and plant height of rice seeds, and is generally superior to the suspension concentrate of cyproconazole.

[0153] Table 7. Effects of functionalized metal nanoparticles on the germination rate of rice seeds

[0154]

[0155] Table 8. Effects of functionalized metal nanoparticles on rice seed germination rate

[0156]

Claims

1. A method for preparing a nano metal skeleton carrier, comprising the following steps: 1) dissolving zirconium tetrachloride in N,N-dimethylformamide, and then adding terephthalic acid to obtain a mixture; 2) Adding glacial acetic acid to the mixture obtained in step 1) for hydrothermal reaction to prepare a nano metal skeleton carrier.

2. The method according to claim 1, characterized in that: In the step 1) and the step 2), the mass ratio of zirconium tetrachloride, terephthalic acid and glacial acetic acid is (1-2):1:(20-40); In the step 2), the mixture and glacial acetic acid are subjected to a hydrothermal reaction in an oven to prepare a nano-metal skeleton carrier.

3. The nano metal skeleton carrier prepared by the method according to claim 1 or 2.

4. Functionalized nano-metal drug-carrying particles are pesticide particles prepared using the nano-metal skeleton described in claim 3 as a carrier.

5. A method for preparing functionalized metal nanoparticles loaded with drugs, characterized in that: The following steps are involved: 1) dissolving the active ingredient of the pesticide in N,N-dimethylformamide, then adding the nano-metal skeleton carrier according to claim 3, stirring and adsorbing at room temperature after ultrasonication to obtain nano-metal drug-carrying particles; 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.

6. The method according to claim 5, characterized in that In the step 2), the mass ratio of the nano metal skeleton carrier loaded with pesticide, tannic acid, and metal salt is 1:1:(2-4), preferably 1:1:

3.

7. The method according to claim 6, characterized in that The method for modifying with tannic acid and metal salt as the modifying substance in step 2) comprises the following steps: A) mixing the nano-metal skeleton carrier loaded with pesticide and the surfactant solution in a mass ratio of 1:1000, and fully dispersing them by ultrasonication at room temperature to prepare a nano-metal drug-loaded particle dispersion; B) adding an aqueous solution of tannic acid to the dispersion of the nano-metal drug-loaded particles obtained in step A), and then adding an aqueous solution of a metal salt dropwise, stirring and reacting at 1200 rpm for 5 min, vortexing and shaking for 30 min, centrifuging to obtain a precipitate, and washing three times with ethanol to remove unreacted tannic acid and metal salt, thereby obtaining functionalized nano-metal drug-loaded particles. Preferably, in step A), the ultrasonic time is 60 min, and the mass percentage concentration in the surfactant solution is 0.1-6%, preferably 1%; In the step A), the surfactant is OP-10#, 4917#, OP-21#, 1086#, 602# and 3300#, preferably 602#; In the step B), the metal salt is copper sulfate, zinc acetate, or iron sulfate, preferably zinc acetate.

8. Application of tannic acid metal complexes as encapsulation materials for metal drug-loaded nanoparticles in seed treatment.

9. Use of the method according to claim 1 or 2, or the nano metal skeleton carrier according to claim 3, or the method according to any one of claims 5 to 7 in the preparation of nano pesticides.

10. Application of the method according to any one of claims 5 to 7 or the functionalized nano-metal drug-loaded particles prepared therefrom in seed treatment; the seed treatment methods include but are not limited to seed soaking, seed mixing, seed coating and seed pelleting; the seed treatment functions to promote the germination, emergence and growth of rice seeds, and to prevent and control rice diseases and insect pests.

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