A method for preparing carbon-based kasugamycin nanoparticles and their applications

Abstract

This invention relates to a method for preparing a carbon-based kasugamycin nanoparticle formulation and its application. The method uses carbon dioxide nanoparticles as a substrate, grafted with polyethylene glycol to construct a nanocarrier system with good biocompatibility and loading capacity, and then inoculates kasugamycin, providing a carbon-based kasugamycin nanocomposite formulation with slow-release characteristics. This formulation effectively avoids interference from soil organic matter in real soil environments, exhibiting excellent slow-release behavior and enhanced bactericidal efficiency. Sand column simulation experiments showed a bacterial efflux rate of 0%, indicating that KAS / PEG@CNT maintained its high adsorption capacity under leaching conditions (such as rainfall and water spray), capable of capturing all injected bacteria. The stable loading and slow release of kasugamycin in the formulation provides a new technical solution for the long-term and efficient control of animal-derived pathogen contamination in agricultural environments, effectively reducing the risk of agricultural products being contaminated by animal-derived pathogens and the spread of foodborne diseases.

Description

Technical Field

[0001] This invention relates to the field of agricultural biomedicine technology, specifically to a method for preparing carbon-based kasugamycin nanoparticles and their applications. Background Technology

[0002] Animal-derived pathogens (such as Salmonella) can enter the farmland environment through livestock and poultry manure, irrigation water, and soil, contaminating agricultural products and ultimately harming human health through the food chain. Kasugamycin, a widely used aminoglycoside antibiotic in agriculture, exhibits good inhibitory activity against Gram-negative bacteria and has the potential for application in controlling pathogenic microorganisms in farmland environments. However, traditional antibiotics are easily lost through leaching (rainfall, irrigation), with an effective utilization rate typically below 30%, requiring multiple applications. This leads to antibiotic residues and accumulation in the soil environment, posing a potential ecological risk.

[0003] Carbon-based nanomaterials, due to their small size and high specific surface area, are widely used for the adsorption and removal of pollutants (such as pesticides, antibiotics, and heavy metals) from farmland soil. Among them, carbon nanotubes, with their unique tubular structure, easily functionalizable surface, and good charge properties, have become important materials for nanoparticle carrier research. Combining carbon nanotubes with antibiotics holds promise for achieving a synergistic effect of adsorption and bactericidal action.

[0004] However, natural organic matter (such as humus) that is widely present in soil can interact with nanomaterials, occupying adsorption sites, changing their surface properties, and reducing their removal efficiency against target pathogens. Summary of the Invention

[0005] In view of the deficiencies of the prior art, the purpose of this invention is to provide a method for preparing carbon-based kasugamycin nanoparticles and their applications, so as to solve the problems mentioned in the background art.

[0006] The present invention solves the technical problem by adopting the following technical solution: One objective of this invention is to provide a method for preparing carbon-based kasugamycin nanoparticles, comprising the following specific preparation steps: A. Disperse multi-walled carbon nanotubes in concentrated sulfuric acid using ultrasonication, then slowly add concentrated sulfuric acid and concentrated nitric acid dropwise to the dispersion, controlling the reaction solution temperature to room temperature. After the addition is complete, heat the reaction solution in a water bath. After cooling, pour the reaction solution into ice water to dilute it, and repeatedly centrifuge and wash until neutral. Freeze-dry to obtain carbon oxide nanotubes. B. Melt polyethylene glycol, add the above-mentioned carbon oxide nanotubes and concentrated sulfuric acid in sequence. The mass ratio of carbon oxide nanotubes, concentrated sulfuric acid and polyethylene glycol is 1:2~4:8~12. After ultrasonic dispersion, stir and react in a water bath at 80~100℃ under nitrogen atmosphere for 4~8 hours. Wash with petroleum ether, acetone and deionized water in sequence until neutral. Freeze dry and sieve to obtain polyethylene glycol grafted carbon oxide nanotubes. C. Next, ultrasonically disperse polyethylene glycol-grafted carbon oxide nanotubes in distilled water, adjust the pH to 6-8, slowly add kasugamycin solution while stirring, continue stirring, filter, freeze dry, and sieve to obtain carbon-based kasugamycin nano-formulation.

[0007] Further, in step A of the preparation method, each 1g of multi-walled carbon nanotubes is added to 10-100 mL of concentrated sulfuric acid and ultrasonically dispersed for 1-4 hours; the added concentrated sulfuric acid or concentrated nitric acid is calculated at 1g:10-50mL.

[0008] Furthermore, in step A of the preparation method, the water bath heating temperature is 60-90 ℃, and the reaction time is 2-6 hours.

[0009] Furthermore, in step B of the preparation method, the molecular weight of polyethylene glycol is in the range of 1000-2000, and the mass ratio of polyethylene glycol, carbon oxide nanotubes and concentrated sulfuric acid is 1:2~4:8~12.

[0010] Preferably, the mass ratio of polyethylene glycol, carbon nanotubes and concentrated sulfuric acid is 1:3.68:10.

[0011] Further, in step B of the preparation method, after ultrasonic dispersion, the mixture is stirred and reacted in a water bath at 80~100℃ for 4-8 hours under an inert gas atmosphere.

[0012] The inert gas is nitrogen.

[0013] Furthermore, in step C of the preparation method, the mass ratio of polyethylene glycol-grafted carbon oxide nanotubes to kasugamycin is 1:0.25-0.75, and the pH is 7-8.

[0014] Furthermore, in step C of the preparation method, when polyethylene glycol-grafted carbon oxide nanotubes are ultrasonically dispersed, the ultrasonic power is 200~600W and the total ultrasonic time is 20~40 minutes.

[0015] A second objective of this invention is to provide a carbon-based kasugamycin nanoformulation obtained by any of the above preparation methods.

[0016] A third objective of this invention is to provide the application of any of the above preparation methods or carbon-based kasugamycin nanoformulations obtained by the preparation methods in pesticide formulations.

[0017] Compared with existing technologies, this invention has the following advantages: This invention uses multi-walled carbon nanotubes to prepare carbon oxide nanoparticles as the substrate material. First, it is composite-modified with polyethylene glycol to construct a nanocarrier system with good biocompatibility and loading capacity. Then, kasugamycin is introduced via electrostatic adsorption, successfully preparing a carbon-based kasugamycin nanocomposite formulation (KAS / PEG@CNT) with slow-release characteristics. Infrared spectroscopy and thermogravimetric analysis were performed on the morphology and structure of the material. The results showed that kasugamycin was effectively loaded on the surface of the polyethylene glycol-modified carbon oxide nanoparticles, with KAS accounting for approximately 18% of the total drug mass. Sand column experiments showed that the adsorption rate of this formulation for bacteria in soil was similar to that in an environment free of organic matter, indicating that the adsorption of bacteria by this reagent is not affected by soil organic matter. Compared with other materials, the bacterial survival rate in the sand column extract was 66%, reduced by half, indicating that organic matter does not affect the good bactericidal ability of this formulation. The release of kasugamycin by the formulation was slow and stable, with a 38% lower release rate compared to kasugamycin containing KAS@CNT sand columns, significantly prolonging the action time of kasugamycin, indicating that this formulation has good anti-leaching and scouring properties, and the sustained-release effect is not affected by organic matter. In summary, the sustained-release system formed by this formulation not only effectively avoids the bottleneck problem of organic matter interference, but also maintains its long-lasting effectiveness in leaching environments (such as rainwater scouring and irrigation) by improving the sustained-release capacity. This greatly reduces leaching loss and the need for frequent dosing, improves the utilization efficiency of kasugamycin, and reduces the side effects of kasugamycin, providing a new strategy for achieving long-acting, low-toxicity, and highly effective antibacterial treatment of crops. Attached Figure Description

[0018] Figure 1 The infrared spectrum of KAS / PEG@CNT is shown, with infrared spectra of CNT, PEG@CNT and KAS@CNT also shown as controls.

[0019] Figure 2 The particle size distribution of four carbon nanotube-based materials is shown in simulated soil water and real soil water.

[0020] Figure 3 Thermogravimetric analysis (TGA) plots for KAS@CNT and KAS / PEG@CNT.

[0021] Figure 4 The minimum bactericidal concentrations of KAS@CNT and KAS / PEG@CNT against Salmonella typhimurium are given.

[0022] Figure 5 The release rates of kasugamycin in sand columns encapsulated with KAS, KAS@CNT and KAS / PEG@CNT under different background solution rinsing conditions were determined.

[0023] Figure 6 This is a diagram of a bacterial migration experimental setup.

[0024] Figure 7 Breakthrough curves for bacterial migration in sand columns containing different packing materials.

[0025] Figure 8 The number and survival rate of surviving bacteria in the sand column extract (which was allowed to stand for 3 hours to fully sterilize) and the column end effluent.

[0026] Figure 9 The image shows a scanning electron microscope (SEM) image of KAS / PEG@CNT, with infrared spectra of CNT, PEG@CNT, and KAS@CNT shown as controls. Detailed Implementation

[0027] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings and specific examples. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] abbreviation: CNT: Carbon nanotube KAS: Kasugamycin PEG: Polyethylene glycol A method for preparing a carbon-based kasugamycin nanoparticle formulation (KAS / PEG@CNT) includes the following steps: 1. Add 1 g of multi-walled carbon nanotubes to 10-100 mL of concentrated sulfuric acid (98% by mass) and ultrasonically disperse for 1-4 hours at 600 W, preferably 50 mL of concentrated sulfuric acid and 2 hours of ultrasonic dispersion. Measure 10-50 mL of concentrated sulfuric acid and 10-50 mL of concentrated nitric acid (68% by mass), preferably 25 mL of concentrated sulfuric acid and 25 mL of concentrated nitric acid. Slowly add the sulfuric acid first, then the nitric acid, dropwise to the dispersion. Use an ice bath to control the system temperature at room temperature (20-25°C). After the addition is complete, transfer the system to a water bath and heat to 60-90°C, preferably 70°C, for 2-6 hours, preferably 4 hours. After cooling, dilute the reaction solution in 800 mL of ice water (4-10 times the volume of the reaction solution), repeatedly centrifuge and wash until neutral, freeze-dry at -80°C, and obtain carbon nanotubes (CNTs) by sieving through a 50-80 mesh sieve.

[0029] 2. Polyethylene glycol (average molecular weight ~1000-2000 Da) is melted at 90℃, preferably with a molecular weight of 1000 Da. The aforementioned carbon oxide nanotubes and concentrated sulfuric acid are added sequentially. The mass ratio of carbon oxide nanotubes, concentrated sulfuric acid, and polyethylene glycol is 1:2~4:8~12, preferably 1:3.68:10. After ultrasonic dispersion for 20~40 minutes, the mixture is stirred and reacted in a water bath at 80-100℃ under a nitrogen atmosphere for 4-8 hours, preferably at 90℃ for 6 hours. The mixture is washed sequentially with petroleum ether, acetone, and deionized water until neutral, freeze-dried, and sieved to obtain 50-80 mesh (180-270 μm) material particles, yielding polyethylene glycol-grafted carbon oxide nanotubes (PEG@CNT).

[0030] 3. Take 1 g of polyethylene glycol-grafted carbon oxide nanotubes and ultrasonically disperse them in 500 mL of distilled water. The ultrasonic power is 300 W, with a working time of 1-3 seconds and an intermittent time of 1-3 seconds, for a total ultrasonic time of 20-40 minutes, preferably 30 minutes. Adjust the pH to 7-8, preferably 7.5. Slowly add 500 mL of a 0.5-1.5 g / L kasugamycin solution with a pH of 7-8 while stirring at 10000 rpm. Preferably, the kasugamycin concentration is 1 g / L and the pH is 7.5. Continue stirring for 30 minutes. Filter, freeze-dry, and sieve out 50-80 mesh material particles (all sieves described below are 50-80 mesh) to obtain the carbon-based kasugamycin nano-formulation KAS / PEG@CNT.

[0031] Example 1: Preparation of a carbon-based kasugamycin nanoformulation (KAS / PEG@CNT) The steps are as follows: 1. Weigh 2 g of multi-walled carbon nanotubes and add them to 100 mL of concentrated sulfuric acid. Disperse by sonication for 2 hours. Measure 50 mL of concentrated sulfuric acid and 50 mL of concentrated nitric acid and slowly add them dropwise to the dispersion, maintaining the system temperature at room temperature (20-25℃). After the addition is complete, heat the mixture to 70℃ in a water bath and react for 4 hours. After cooling, dilute the reaction solution in 800 mL of ice water, repeatedly centrifuge and wash until neutral, then freeze-dry at -80℃ for 48 hours. Separate the solution through a 50-80 mesh sieve to obtain carbon nanotubes (CNTs).

[0032] 2. Polyethylene glycol (molecular weight 1000-2000) was melted at 90℃, and the above-mentioned carbon oxide nanotubes and concentrated sulfuric acid were added sequentially. The mass ratio of carbon oxide nanotubes, concentrated sulfuric acid and polyethylene glycol was 1:3.68:10. After ultrasonic dispersion for 30 minutes, the mixture was stirred and reacted in a water bath at 90℃ under a nitrogen atmosphere for 6 hours. The mixture was washed sequentially with petroleum ether, acetone and deionized water until neutral, freeze-dried, and sieved to obtain 50-80 mesh (180-270μm) material particles to obtain polyethylene glycol-grafted carbon oxide nanotubes (PEG@CNT).

[0033] 3. Take 0.2 g of polyethylene glycol-grafted carbon oxide nanotubes and ultrasonically disperse them in 100 mL of distilled water. The ultrasonic power is 300 W, with a working time of 1 second and an intermittent time of 1 second, for a total ultrasonic time of 30 minutes. Adjust the pH to 7.5, and slowly add 100 mL of kasugamycin solution with a concentration of 1 g / L and a pH of 7.5 while stirring at 800 rpm. Continue stirring for 30 minutes. Filter, freeze-dry, and sieve using a 50-80 mesh sieve to obtain the carbon-based kasugamycin nanoformulation KAS / PEG@CNT.

[0034] Comparative Example 1: Preparation of Carbon Oxide Nanotubes (CNTs) The steps are as follows: Weigh 2 g of multi-walled carbon nanotubes and add them to 100 mL of concentrated sulfuric acid. Disperse the mixture by sonication for 2 hours. Measure 50 mL of concentrated sulfuric acid and 50 mL of concentrated nitric acid and slowly add them dropwise to the dispersion. Maintain the system temperature at room temperature. After the addition is complete, raise the temperature to 70 °C and react for 4 hours. After cooling, dilute the reaction solution in 800 mL of ice water, centrifuge and wash repeatedly until neutral, freeze-dry at -80 °C for 48 hours, and sieve to obtain carbon nanotubes (CNTs).

[0035] Comparative Example 2: Preparation of polyethylene glycol-grafted carbon oxide nanotubes (PEG@CNT) The steps are as follows: The carbon oxide nanotubes prepared in Comparative Example 1 were subjected to a polyethylene glycol grafting reaction according to the method in Example 1. They were washed with petroleum ether, acetone and deionized water until neutral, freeze-dried and sieved to obtain polyethylene glycol-grafted carbon oxide nanotubes (PEG@CNT).

[0036] Comparative Example 3: Preparation of carbon oxide nanotubes (KAS@CNT) loaded with kasugamycin Take 0.2 g of carbon oxide nanotubes prepared in Comparative Example 1, disperse them in 100 mL of distilled water, and sonicate them for 30 minutes with an ultrasonic power of 300 W, working for 1 second and then pausing for 1 second. Adjust the pH to 7.5, and slowly add 100 mL of kasugamycin solution with a concentration of 1 g / L and a pH of 8 while stirring at 800 rpm. Continue stirring for 30 minutes. Filter, freeze dry, and sieve through a 50-80 mesh to obtain carbon oxide nanotubes loaded with kasugamycin (KAS@CNT).

[0037] Example 2

[0038] 1. Infrared spectroscopy testing of carbon-based kasugamycin nanoparticles Infrared spectroscopy analysis of the nanoformulations was performed on an Agilent Technologies Cary 630 FT-IR spectrometer. The wavenumber range was 400–4000 cm⁻¹. -1Experimental procedure: When performing FT-IR testing, weigh out the amounts of potassium bromide (m) that has been freeze-dried at -80 ℃ from Examples 1 and Comparative Examples 1-3, respectively. 样品 :m 溴化钾 =1:300), place in an agate mortar and mix thoroughly, then grind thoroughly and press into tablets for testing.

[0039] Figure 1 Fourier transform infrared (FT-IR) spectra of four carbon-based materials are shown. The results indicate that, compared to CNTs, KAS@CNTs exhibit superior performance in the 1150-1040 cm⁻¹ range. -1 The region shows obvious absorption peaks for COC and C-OH, and at 3449 cm⁻¹ -1 The enhanced hydroxyl peak at 2926 and 2858 cm⁻¹ indicates that the glycoside structure and hydroxyl groups of kasugamycin were successfully introduced into the CNT surface. PEG@CNT, on the other hand, showed peaks at 2926 and 2858 cm⁻¹. -1 A typical methylene stretching vibration peak appears, and it is at 1100 cm⁻¹. -1 Characteristic peaks of PEG ether bonds appeared nearby. KAS / PEG@CNT simultaneously retained characteristic absorption peaks of both PEG and kasugamycin, indicating that both components were successfully modified at the CNT interface.

[0040] 2. Particle size of carbon-based kasugamycin nanoparticles in different background solutions Under ice bath conditions, carbon nanotubes prepared in Example 1 and Comparative Examples 1-3 were dispersed in pure water or real soil water using a cell disruptor at a power of 300W, with a cycle of 1 second on and 1 second off, for a total sonication time of 30 minutes, yielding a carbon nanotube suspension of 1 mg / L. The particle size of the nanomaterials in the suspension was determined using a Malvern laser particle size analyzer and the dynamic light scattering method.

[0041] like Figure 2 As shown, the particle size of CNT, KAS@CNT, PEG@CNT, and KAS / PEG@CNT in real soil water increased by 920%, 949%, 21%, and 22%, respectively, compared to pure water. The particle size of ungrafted PEG carbon nanotubes increased by more than 9 times, indicating that in soil water, the material aggregates due to the interaction between soluble organic matter (adsorption) and multivalent cations such as calcium and magnesium (bonding effect), forming larger aggregates. The PEG-grafted material can inhibit the adhesion of soluble organic matter, with a small increase in particle size, thus exhibiting good stability in real soil water.

[0042] 3. Thermogravimetric analysis of carbon-based kasugamycin nanoformulations Thermogravimetric analysis of Example 1 and Comparative Example 3 was performed using a NETZSCH STA 449 F5 / F3 Jupiter manufactured by NETZSCH GmbH, Germany. The test temperature range was 20-900 °C.

[0043] like Figure 3 The KAS@CNT curve shows only one distinct weight loss phase within the 150-400 ℃ range, with a single thermal decomposition peak corresponding to the DTG curve. The total weight loss of KAS@CNT in this temperature range is approximately 22%, which deviates from the maximum adsorption capacity of CNT for kasugamycin (322 mg / g, 24 wt%) by only about 2%. This indicates that the weight loss measured by TGA mainly originates from the decomposition of kasugamycin, proving that the actual kasugamycin loading in KAS@CNT is approximately 22%. KAS / PEG@CNT exhibits two consecutive weight loss processes within the 150-500 ℃ range, with a total weight loss of approximately 30%. This is due to the thermal decomposition of both PEG and kasugamycin within this temperature range.

[0044] 4. Determination of the minimum bactericidal concentration of carbon-based kasugamycin nano-formulation against Salmonella. Salmonella typhimurium was cultured in beef extract peptone medium at 37 °C and 150 rpm until the logarithmic growth phase. After centrifugation and washing, the culture was resuspended in sterile physiological saline. Kasugamycin, Examples 1, Comparative Examples 1 and 3 were prepared into different concentration gradients (125, 250, 500, 1000, 2000, 2500, 3000, 4000 and 50000 mg / L) using sterile medium. An equal volume of bacterial suspension was added to each group to make the bacterial concentration 10. 6 CFU / mL. A blank control group (without carbon nanotubes) and a material blank group (without bacterial culture) were set up. Each group was incubated at 37 ℃ with shaking at 150 rpm for 24 h. After incubation, the supernatant of each group mixture was used for 10... -6 Up to 10 -8 The solution was serially diluted, and 100 μL of the diluted solution was evenly spread on the surface of a nutrient agar plate. After incubation at 37 °C for 24 h, the number of colonies was counted by plate counting. The lowest material concentration at which no visible colonies grew on the plate was defined as the minimum bactericidal concentration (MBC) of the material.

[0045] according to Figure 4 As shown, CNTs did not exhibit significant bactericidal activity within the experimental concentration range, while the minimum bactericidal concentrations (MBCs) of KAS, KAS@CNT, and KAS / PEG@CNT against Salmonella were 500, 2500, and 3000 mg / L, respectively. Since CNTs themselves do not possess significant bactericidal ability, the bactericidal effect of both composite materials should originate from the kasugamycin loaded on their surfaces.

[0046] For KAS@CNT, its MBC is 2500 mg / L, which is 5 times that of free kasugamycin MBC. This means that to achieve the same bactericidal effect, approximately 20% active kasugamycin (500 / 2500=20%) needs to be contained in a unit mass of KAS@CNT. This value is highly consistent with the 22 wt% measured by TGA and the 24 wt% obtained by adsorption experiments, indicating that the kasugamycin in KAS@CNT basically maintains its biological activity, and its bactericidal ability comes entirely from the kasugamycin loaded on the surface.

[0047] For KAS / PEG@CNT, its MBC is 3000 mg / L, which is 6 times that of free kasugamycin. Therefore, at least approximately 16.7% effective kasugamycin (500 / 3000=16.7%) should be present per unit mass of material to achieve complete sterilization. Since PEG itself does not have bactericidal activity, this effective kasugamycin content can be considered as the lower limit of the actual kasugamycin loading in the material. During the material preparation process, the maximum adsorption capacity of PEG@CNT for kasugamycin was measured to be 224 mg / L (18 wt%), which is highly consistent with the 16.7 wt% obtained by back-calculation of MBC. Therefore, the actual kasugamycin loading in KAS / PEG@CNT is approximately 18 wt%.

[0048] 5. Scanning electron micrograph of carbon-based kasugamycin nanoformulation The morphology of Examples 1 and Comparative Examples 1-3 was observed using a Regulus 8100 field emission scanning electron microscope manufactured by Hitachi, Ltd., Japan. During testing, to ensure good conductivity, a small amount of sample was evenly coated with conductive adhesive and then sputter-coated with gold.

[0049] according to Figure 9The basic framework structure of carbon nanotubes (CNTs) in four materials—CNT, KAS@CNT, PEG@CNT, and KAS / PEG@CNT—was observed to be intact, without significant large-scale aggregation or structural collapse. The average tube diameters of the four samples were 20±4 nm, 20±6 nm, 24±6 nm, and 25±5 nm, respectively. The CNT surface was smoother than the tube body surface, with a uniform tube diameter distribution. Slight granular or film-like deposits were observed on the surface of some KAS@CNT nanotubes. The tube diameter of PEG@CNTs was thicker, and a large number of obvious encapsulated particles were observed on the surface, with relatively uniform encapsulation. The KAS / PEG@CNT surface also had a large number of encapsulated particles, and was rougher than the PEG@CNT surface. These results indicate that, while maintaining the integrity of the CNT tubular structure, the introduction of KAS and PEG both led to different degrees of changes in the CNT surface morphology. PEG modification had a more significant covering effect on the tube body surface, while the synergistic modification of KAS and PEG further increased the surface roughness of the material, providing a good structural basis for the subsequent stable loading of active components and the improvement of material performance.

[0050] Example 3: Kasugamycin sustained-release capability of carbon-based kasugamycin nanoformulation (KAS / PEG@CNT) Following the method described in Example 1, kasugamycin KAS, KAS@CNT, and KAS / PEG@CNT were mixed with iron oxide quartz sand at mass ratios of 1:82, 1:21, and 1:16, respectively, and packed into chromatography columns to ensure that the total amount of kasugamycin in each mixed sand column was 45 mg. Real soil water was injected from the bottom of the chromatography column at a flow rate of 1 mL / min. When liquid flowed from the top of the sand column, the column was immediately inverted to inject background solution from top to bottom, and the eluent was collected every 10 minutes. After filtering the eluent through a 0.45 μm filter membrane, the concentration of kasugamycin was determined by high-performance liquid chromatography (HPLC). A bar graph was plotted with collection time on the x-axis and the kasugamycin content in the eluent on the y-axis to analyze the release of kasugamycin from the mixed sand.

[0051] High-performance liquid chromatography (HPLC): Kasugamycin standard was weighed and prepared into standard solutions with mass concentrations of 0, 5, 10, 20, 50, and 100 mg / L using ultrapure water to establish a standard curve. The liquid samples to be tested were filtered through a 0.22 μm nylon membrane before use. An HPLC system equipped with a UV detector was used, with an Aqua C18 reversed-phase column (250 mm × 4.6 mm, 5 μm). Mobile phase A was 0.067 mol / L phosphate buffer (pH 7.0), and mobile phase B was methanol, with a volume ratio of 1:1. The flow rate was 1.0 mL / min, the column temperature was 30 ℃, the detection wavelength was 210 nm, and the injection volume was 20 μL. A standard curve was established with the standard solution concentration as the x-axis and the peak area as the y-axis. The concentration of kasugamycin in the liquid samples was calculated using the external standard method.

[0052] like Figure 5 As shown, the release of kasugamycin in all materials exhibited a rapid release pattern in the first 10-20 minutes, followed by a slowdown from 20-60 minutes. The kasugamycin release was fastest in the kasugamycin-containing mixed sand (KAS), with 98% released within the first 10 minutes, followed by almost no release. Under background solution rinsing, the kasugamycin release rate of the KAS@CNT-containing mixed sand column was 47%, significantly lower than that of the kasugamycin-containing mixed sand column. The kasugamycin release rate of the KAS / PEG@CNT-containing mixed sand column in the background solution was 32%, further decreasing compared to the kasugamycin-containing mixed sand column, and 38% lower than that of the KAS@CNT-containing sand column, indicating that KAS / PEG@CNT has good anti-leaching properties.

[0053] Example 4: Bacterial Retention Capacity of Carbon-Based Kasugamycin Nanoparticles (KAS / PEG @CNT) 1. Packing material preparation The filler used in the experiment was iron oxide-coated quartz sand and carbon-based kasugamycin nanoparticles or a mixture of three other types of carbon nanotubes.

[0054] Synthesis of iron oxide-coated quartz sand (hereinafter referred to as iron oxide sand): 500 g of quartz sand with a particle size of 50-80 mesh was soaked in 1 L of 6 mol / L hydrochloric acid for 48 hours, repeatedly rinsed with distilled water until the pH of the rinsing solution was 7, and dried at 100℃ to constant weight. 500 mL of 1.5 mol / L FeCl3 solution was added to the sand, and it was ultrasonically dispersed for 30 minutes. Excess solution was filtered off, and it was air-dried at 35℃ for 3 hours. The dried quartz sand was soaked in 500 mL of 3 mol / L ammonia water, ultrasonicated for 30 minutes, excess solution was filtered off, and it was dried at 60℃ overnight. It was rinsed with distilled water until the absorbance of the supernatant of the rinsing solution was constant at a wavelength of 600 nm. The 50-80 mesh iron oxide sand was sieved out, autoclaved at 121℃ for 20 minutes, and cooled to room temperature for later use.

[0055] The materials prepared in Example 1 and Comparative Examples 1-3 were sieved to obtain 50-80 mesh particles. Sterilized iron oxide sand was mixed with the sieved material particles at a mass ratio of 17:1, and pure kasugamycin was used as a control. The mixture was shaken at 300 rpm / min for 24 hours in the dark at 5 °C to obtain a mixed filler containing bactericidal materials or nanoparticles.

[0056] 2. Background solution preparation The background solution used in the experiment was soil water containing soluble soil organic matter and salts, obtained by rinsing real soil.

[0057] Soil samples were collected from the campus of Shenyang University (41.832376°N, 123.466847°E) at a depth of 25 to 30 cm. The samples were air-dried in a cool, well-ventilated place for 7 days, and then passed through a 10-mesh sieve to remove large stones and tree roots. A certain amount of soil sample was weighed and mixed with ultrapure water at a soil-to-water ratio of 1:5, and shaken for 24 hours. The suspension was centrifuged at 8000 rpm, and the supernatant was filtered through a 0.45 μm sterile filter membrane. The resulting filtrate was the actual soil aqueous solution.

[0058] 3. Preparation of bacterial culture Salmonella typhimurium (S. typhimurium 50115) was purchased from the China Medical Bacterial Culture Collection Center. The strain was inoculated onto beef extract peptone medium and cultured at 37 ℃ and 150 rpm until the logarithmic growth phase. The bacterial cells were isolated using a benchtop high-speed centrifuge at 8000 rpm, resuspended in background solution, and washed three times. The OD of the bacterial suspension was adjusted at 600 nm using a UV-Vis spectrophotometer. 600 Up to 0.015 (1.6 × 10 7 (CFU / mL), used as the working solution for bacterial migration experiments.

[0059] 4. Bacterial migration experiment like Figure 6As shown, the migration experimental system consists of an injection device, an iron oxide sand-packed column, and an effluent detection and collection device. The injection device includes a sample vial and a peristaltic pump. The porous media column consists of a chromatography column with an inner diameter of 1 cm and a length of 10 cm, and the mixed packing material inside. In a clean bench, the mixed packing material is wet-filled into the chromatography column to a height of 3 cm, forming a sand column with a porosity of 0.42 and a pore volume of 0.99 mL. 50 PV of background solution is injected from the bottom of the chromatography column at a flow rate of 1 mL / min to equilibrate the system. After equilibration, the chromatography column is inverted, and the working solution is injected from the top to begin the bacterial migration experiment: first, 10 PV of background solution is injected at a flow rate of 1 mL / min, then 10 PV of bacterial solution is injected, and finally, the flow rate is switched back to the background solution to continue injecting 50 PV until the effluent OD... 600 The value was lowered to baseline. The absorbance of the effluent was directly measured at 600 nm using a UV spectrophotometer. A breakthrough curve was plotted with the pore volume (PV) of the injected solution on the x-axis and the ratio of the bacterial concentration in the effluent to the bacterial concentration in the injected solution (C / C0) on the y-axis.

[0060] Figure 7 Breakthrough curves of Salmonella in iron oxide sand columns mixed with different materials are presented. The efflux rates of Salmonella in iron oxide sand, KAS-mixed iron oxide sand, CNT-mixed iron oxide sand, KAS@CNT-mixed iron oxide sand, PEG@CNT-mixed iron oxide sand, and KAS / PEG@CNT-mixed iron oxide sand were 80%, 77%, 50%, 53%, 68%, and 0%, respectively. The results indicate that KAS / PEG@CNT exhibited the best bacterial retention performance among the tested materials. Under soil water conditions, no Salmonella efflux was detected in the KAS / PEG@CNT mixed sand column, with an efflux rate of 0%, achieving complete capture of the injected bacteria. In contrast, the other control systems all showed varying degrees of bacterial efflux, with efflux rates ranging from 50% to 80%. In conclusion, in real soil water containing soluble organic matter, KAS / PEG@CNT can significantly inhibit the migration of Salmonella in sand columns and maintain excellent bacterial capture ability, demonstrating a significantly better bacterial adsorption effect than the control systems 1-3.

[0061] Example 5: Bactericidal effect of carbon-based kasugamycin nano-formulation (KAS / PEG @CNT) Collect the sand column effluent from the 3rd to 13th PV after bacterial inoculation in Example 3. After the bacterial migration experiment, let the sand column effluent and sand column stand together at room temperature in the dark for 2 hours. Then, in a clean bench, take 0.45 g of mixed sand from the top of the sand column, place it in a centrifuge tube, dilute it 100 times with background solution, vortex for 3 minutes, let it stand for 1 minute, and take the supernatant of the sand column extract and perform 10-fold filtration with background solution. -2 Up to 10-4 Perform serial dilutions. Take another 10-fold serial dilution of the supernatant from the effluent. -4 Up to 10 -6 Serial dilutions were performed. Bacterial counting was conducted on each dilution using the plate plating method, and the bacterial concentration (Cb) of the sand column extract was measured. Sexp and the bacterial concentration C of the sand column effluent Eexp Take 1 mL of the initial bacterial suspension, dilute appropriately, and count the bacteria using the same method to obtain the initial bacterial concentration C. Oexp .

[0062] Based on the background solution and bacterial suspension injection volume and sequence of the bacterial migration experiment in Example 4, the bacteria were injected sequentially without passing through a chromatography column, and the OD was measured using a UV spectrophotometer. 600 The trend of value change is for the control group without medium.

[0063] The area S of the experimental breakthrough curve was obtained through integral calculation. St and the breakthrough curve area S of the control group without media Ot The concentration C of bacteria captured within the sand column was calculated using equations (1) and (2). St and the bacterial concentration C of the outflowing sand column Et .

[0064] (1) (2) Based on equations (3) and (4), the bacterial survival rates in the sand column and in the effluent were calculated 3 hours after bacterial injection.

[0065] (3) (4) Figure 8 The study presented the survival rate and quantity of bacteria in sand column extracts and effluents containing different nano-formulations 3 hours after bacterial injection. Results showed that the bacterial survival rates in the sand column extracts of iron oxide sand, kasugamycin-mixed sand, CNT-mixed sand, and PEG@CNT-mixed sand all exceeded 130%. The bacterial survival rate in the KAS@CNT-mixed sand column was 117%, indicating that CNT, PEG@CNT, and KAS@CNT failed to effectively remove bacteria in real soil water, and the bacteria even multiplied. In contrast, the bacterial survival rate in the sand column extract of KAS / PEG@CNT-mixed sand was 66%, demonstrating that KAS / PEG@CNT possesses good bactericidal ability in real soil water, and no bacteria were detected in the sand column effluent.

[0066] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. A method for preparing a carbon-based kasugamycin nanoparticle formulation, characterized in that, The preparation steps include the following: A. Disperse multi-walled carbon nanotubes in concentrated sulfuric acid using ultrasonication, then slowly add concentrated sulfuric acid and concentrated nitric acid dropwise to the dispersion, controlling the reaction solution temperature to room temperature. After the addition is complete, heat the reaction solution in a water bath. After cooling, pour the reaction solution into ice water to dilute it, and repeatedly centrifuge and wash until neutral. Freeze-dry to obtain carbon oxide nanotubes. B. Melt polyethylene glycol, add the above-mentioned carbon oxide nanotubes and concentrated sulfuric acid in sequence. The mass ratio of carbon oxide nanotubes, concentrated sulfuric acid and polyethylene glycol is 1:2~4:8~12. After ultrasonic dispersion, stir and react in an inert gas atmosphere in a water bath at 80~100 ℃ for 4~8 hours. Wash with petroleum ether, acetone and deionized water in sequence until neutral. Freeze dry and sieve to obtain polyethylene glycol grafted carbon oxide nanotubes. C. Next, ultrasonically disperse polyethylene glycol-grafted carbon oxide nanotubes in distilled water, adjust the pH to 6-8, slowly add kasugamycin solution while stirring, continue stirring, filter, freeze dry, and sieve to obtain carbon-based kasugamycin nano-formulation.

2. The preparation method according to claim 1, characterized in that, In step A, each 1g of multi-walled carbon nanotubes is added to 10-100 mL of concentrated sulfuric acid and ultrasonically dispersed for 1-4 hours; the added concentrated sulfuric acid or concentrated nitric acid is calculated as 1g: 10-50mL.

3. The preparation method according to claim 1, characterized in that, In step A, the water bath heating temperature is 60-90℃, and the reaction time is 2-6 hours.

4. The preparation method according to claim 1, characterized in that, In step B, the molecular weight of polyethylene glycol ranges from 1000 to 2000, and the mass ratio of polyethylene glycol, carbon nanotubes, and concentrated sulfuric acid is 1:2~4:8~12.

5. The preparation method according to claim 1, characterized in that, In step B, after ultrasonic dispersion, the mixture is stirred and reacted in an inert gas atmosphere in a water bath at 80~100℃ for 4-8 hours.

6. The preparation method according to claim 1, characterized in that, In step C, the mass ratio of polyethylene glycol-grafted carbon oxide nanotubes to kasugamycin is 1:0.25-0.75, and the pH is 7-8.

7. The preparation method according to claim 1, characterized in that, In step C, when polyethylene glycol-grafted carbon oxide nanotubes are ultrasonically dispersed, the ultrasonic power is 200~600W and the total ultrasonic time is 20~40 minutes.

8. The preparation method according to claim 1, characterized in that, The sieve is 50-80 mesh.

9. The carbon-based kasugamycin nanoformulation obtained by the preparation method according to any one of claims 1 to 8.

10. The preparation method according to any one of claims 1 to 8 or the application of the carbon-based kasugamycin nano-formulation according to claim 8 in pesticide formulations.

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