Preparation method of ROS-responsive functionalized starch-based drug-loaded nano / microspheres and their application in external defense and internal regulation

Abstract

The present invention belongs to the field of pesticide formulations and relates to a method for preparing a ROS (reactive oxygen species)-responsive starch-based drug-loaded nano-delivery system and its application in achieving "external prevention and internal regulation" of major economically valuable forest pests and diseases. The method for preparing ROS-responsive functionalized starch-based drug-loaded nano / microspheres of the present invention comprises the following steps: preparing a ROS-responsive starch-based wall material PDSE-CMS using carboxymethyl starch; separately preparing an aqueous phase and an oil phase: the aqueous phase contains the ROS-responsive starch-based wall material PDSE-CMS, and the oil phase contains a pesticide and a cross-linking agent; after mixing the oil and aqueous phases, preparing an O / W emulsion, which is then heated for cross-linking and solidification to obtain ROS-responsive functionalized starch-based drug-loaded nano / microspheres. The method of the present invention can achieve "external prevention and internal regulation" prevention and control of apple tree rot from the inside out.

Description

Technical Field

[0001] The present invention belongs to the field of pesticide formulations and relates to a preparation method of a ROS (reactive oxygen species)-responsive starch-based drug-loaded nano-delivery system and its application in achieving "external prevention and internal regulation" of major forest diseases and insect pests with high economic value. Background Art

[0002] Perennial economic trees, such as apple trees, pear trees, grape trees, and bayberry trees, once infected with pathogens, can easily cause the trees to rot and die, causing serious damage. For example: apple trees ( Malus domestica Borkh; Rosaceae), whose fruits are rich in natural antioxidants; however, apple rot (the pathogen is mainly Ascomycota fungi) Valsa mali ) will cause serious economic losses. In view of the severity of its damage, many scholars have studied the pathogenic fungi. Valsa mali The pathogenic mechanism of the disease that attacks apple trees has been studied in detail. Valsa mali This weakly parasitic fungus invades through wounds on the trunk, branches, or twigs, particularly through frostbite and mechanical damage from pruning. Once established, the pathogen rapidly proliferates under favorable conditions and spreads to other areas, causing symptoms such as bark ulcers and tissue necrosis. In severe cases, it can even cause the death of the entire tree. Valsa mali The pathogen's destructive power stems from its ability to secrete large amounts of toxins in the early stages of infection, rapidly killing host cells and promoting colonization. Furthermore, its necrotrophic properties allow it to utilize dead host cells as a nutrient source, aiding its rapid expansion and further infection. Valsa mali The mycelium can not only colonize the cortical parenchyma cells and phloem tissue, but can even invade the xylem. This deep infection characteristic makes it difficult for traditional fungicides (such as methyl thiophanate, tebuconazole and difenoconazole) to effectively penetrate into the plant tissue through the bark, making it impossible to completely eliminate the pathogen. In addition, Valsa mali It also has the characteristic of latent infection, meaning the pathogen can be detected even in uninfected branches. Therefore, traditional control measures, mainly spraying or applying fungicides, are generally only effective against the pathogen on the tree surface, but have little effect on the mycelium and dormant cells hidden within the plant tissues.

[0003] Despite this, chemical control remains the primary forest control strategy due to its rapid effectiveness and high efficacy. This approach primarily involves manually scraping away the diseased tissue and then spraying or applying a high-concentration fungicide suspension or paste for localized treatment from the outside in. However, this method can only inhibit pathogens that come into direct contact, but it is difficult to completely eliminate the mycelium hidden within the vascular tissue. Consequently, existing fungicides have limited effectiveness in inhibiting the spread of established lesions, and the procedures are complex, consuming significant labor and time costs. Therefore, there is an urgent need to identify or develop new technologies that can efficiently deliver fungicides to the interior of plant tissues to fully unleash their antibacterial effects and thus reduce the difficulty of managing apple orchards.

[0004] The rapid development of nanotechnology in recent years has been crucial for developing highly effective and safe new pesticide formulations and achieving sustainable agricultural development. It can address the limitations of traditional pesticides during use. The process of achieving sustained and controlled release of pesticides in nanoparticles requires comprehensive consideration of multiple factors, including the chemical properties of the pesticide, the safety and environmental friendliness of the materials, the suitability of the preparation process, and the cost and economic viability of the materials.

[0005] Within existing functionalized formulation systems, research and development of environmentally friendly and biodegradable delivery materials is increasing, particularly starch-based modified materials. Starch is a natural biomacromolecule that is abundant, inexpensive, biodegradable, biocompatible, non-toxic, and environmentally friendly. It has found widespread application in food, cosmetics, papermaking, plastics, textiles, and pharmaceuticals. Amylose and amylopectin, the primary components of starch, contain numerous hydroxyl groups, which give them excellent hydrophilicity. With the continued advancement of nanomaterial research, starch nanoparticles have garnered significant attention. Starch nanoparticles can be used in biomedical applications such as intravenous drug delivery, drug delivery, and drug delivery. However, the strong hydrophilicity of starch can lead to aggregation. Furthermore, hydrophilic starch nanoparticles are not suitable for encapsulating and delivering hydrophobic drugs. Therefore, hydrophobic modification of natural starch to prepare monodisperse and stable starch nanoparticles has attracted research interest from both the scientific and industrial communities. To date, various methods have been employed to prepare hydrophobically modified starch nanoparticles. For example, propylated starch nanoparticles were prepared using solvent emulsification / diffusion technology; octenylsuccinate-modified starch nanoparticles were prepared using an ionic liquid / oil microemulsion method; long-chain fatty acid-modified starch nanoparticles were prepared using dialysis; and acetylated starch nanoparticles were prepared using nanoprecipitation. These hydrophobically modified starch nanoparticles have been used for the encapsulation and delivery of hydrophobic drugs. Compared to microemulsion and dialysis methods, nanoprecipitation offers advantages such as high yield and ease of purification, making it an economical and time-saving method. While there are many types of hydrophobically modified starch nanoparticles, the hydrophobic groups used to modify starch are primarily propyl, acetyl, and long-chain fatty acyl groups. Patent CN 114957733B discloses Boc-phenylalanine-modified starch nanoparticles, their preparation method, and their application in hydrophobic drug loading. After hydrophobic modification, the drug is loaded via self-assembly. Conventional hydrophobically modified starch drug loading simply improves the starch's hydrophilicity, resulting in more stable or sustained drug delivery, but rarely offers other synergistic enhancements.

[0006] There are also reports on the direct use of sodium carboxymethyl starch (CMS), a widely available, highly hydrophilic natural polysaccharide, in pharmaceutical preparations. For example, CN 112336697 B discloses the use of sodium carboxymethyl starch as a disintegrant in a simvastatin pulse-release tablet. The disintegrant is a common carrier that also has the property of swelling and disintegrating in water.

[0007] CN102406940B discloses the application of nano-scale flaky sodium carboxymethyl starch in a solid dispersion. The nano-scale flaky sodium carboxymethyl starch is used as a carrier and is directly mixed to obtain a mixture of a drug-carrying system, which has a certain drug-carrying and delivery function.

[0008] In their long struggle against pathogens, host plants have evolved a complex set of defense strategies, primarily encompassing innate and adaptive immunity. When plants recognize pathogen-associated molecular patterns (PAMPs), one of the earliest responses is the rapid generation of reactive oxygen species (ROS). ROS, including superoxide, hydroxyl radicals, and singlet oxygen, not only directly cause oxidative damage to pathogens but also serve as signaling molecules to trigger downstream defense responses. To further prevent pathogen invasion, the ROS burst often leads to a local hypersensitive response (HR), a programmed cell death in healthy plant tissues. However, HR is limited against necrotrophic pathogens, which naturally derive nutrients from necrotizing host cells. The antioxidant system within the plant defense system is crucial for maintaining stable intracellular ROS levels. Salicylic acid (SA) is a key regulator of this process and acts as a signaling molecule to induce systemic acquired resistance (SAR). In nanodelivery technologies, ROS are often used as biostimulants to control the release rate of active ingredients from carriers, while the delivery of plant signaling molecules (such as salicylic acid) as plant growth regulators is also common. However, no research has yet synergistically integrated these two strategies to enhance the pest control efficacy of pesticides. Summary of the Invention

[0009] The technical problem to be solved by the present invention is to provide a preparation method of ROS-responsive functionalized starch-based drug-loaded nano / microspheres and their application in external defense and internal regulation.

[0010] In order to solve the above problems, the present invention provides a method for preparing starch-based drug-loaded nano / microspheres with ROS response functionalization (i.e., a method for preparing starch-based drug-loaded sustained-release nano / microspheres with ROS response and oxidation regulation functions), comprising the following steps:

[0011] 1) Carboxymethyl starch (CMS) was used to prepare ROS-responsive starch-based wall material PDSE-CMS;

[0012] 2) Prepare the water phase and oil phase separately:

[0013] The aqueous phase contains ROS-responsive starch-based wall material PDSE-CMS, and the oil phase contains pesticides (fat-soluble pesticides) and a cross-linking agent;

[0014] 3) After the oil phase and the water phase are mixed, an O / W emulsion is prepared, which is then heated for cross-linking and curing (interfacial polymerization reaction) to obtain ROS-responsive functionalized starch-based drug-loaded nano / microspheres (i.e., starch-based polymer sustained-release nano / microspheres loaded with pesticides).

[0015] As an improvement to the preparation method of the ROS-responsive functionalized starch-based drug-loaded nano / microspheres of the present invention:

[0016] 1) Carboxymethyl starch (CMS) was added (dispersed) in a phosphate buffer solution (pH 6.0, 0.01 M). An activator was then added and stirred at 40 ± 10°C for 1–5 h (to activate the carboxyl groups on the CMS molecular chain). PDSE, a ROS-responsive switch, was then added and stirred at 20–80°C (preferably 50 ± 10°C) for 24 ± 1 h. The ROS-responsive starch-based wall material, PDSE-CMS, was obtained as a pale yellow powder after dialysis (molecular weight cut-off: 5000 Da) and freeze-drying.

[0017] The activator consists of EDC and NHS;

[0018] Carboxymethyl starch (CMS): activator: PDSE = 0.1-1.5:0.1-5.0:0.1-10.0 weight ratio (preferably 1.5:1-1.2:4.8-5.2, more preferably 1.5:1.1:5);

[0019] EDC:NHS = 0.1~6:0.1~5.0 weight ratio (preferably 5.5~6:4.5~5, more preferably 6:5);

[0020] EDC is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, NHS is N-hydroxysuccinimide;

[0021] PDSE is 2, 2′-(propane-2, 2-diylbis(sulfonamide))diethylamine;

[0022] Note: For every 1.5 g of carboxymethyl starch (CMS), use 50 ± 10 ml of phosphate buffer solution;

[0023] PDSE is widely used in the construction of ROS-responsive carriers due to the oxidation-sensitive thioketal bond in its structure. It is coupled to the carboxymethyl starch (CMS) backbone through an amide reaction to obtain ROS-responsive starch-based wall materials.

[0024] 2) Dissolving the ROS-responsive starch-based wall material PDSE-CMS obtained in step 1) in an aqueous solution containing a surfactant as the aqueous phase; in the aqueous phase, the concentration of the ROS-responsive starch-based wall material PDSE-CMS is 0.1-5.0 g / 100 ml (preferably 0.1-0.2 g / 100 ml), and the concentration of the surfactant in the aqueous solution containing the surfactant is 0.2-5.0 g / 100 ml (preferably 0.4-0.6 g / 100 ml);

[0025] A pesticide (a fat-soluble pesticide) and 2, 4, 6-tris(4-formylphenoxy)-1, 3, 5-triazine (TFPT) as a cross-linking agent are dissolved in an organic solvent as an oil phase; the mass ratio of the pesticide to the cross-linking agent is 1 to 10:1 (preferably 1 to 5:1); and the amount ratio of the organic solvent to the pesticide in the oil phase is (10 to 80) mL: 1 g.

[0026] 3) The oil phase obtained in step 2) is added to the aqueous phase obtained in step 2) at a volume ratio of organic solvent to aqueous solution containing a surfactant of 1:50-100. After emulsification (high shear emulsification) and cell disruption and dispersion, an O / W emulsion is obtained. The emulsion is then heated for cross-linking and curing to perform an interfacial polymerization reaction to obtain ROS-responsive functionalized starch-based drug-loaded nano / microspheres (i.e., pesticide-loaded starch-based polymer sustained-release nano / microspheres).

[0027] As a further improvement to the preparation method of the ROS-responsive functionalized starch-based drug-loaded nano / microspheres of the present invention, in step 3):

[0028] The emulsification (high shear emulsification) is: emulsification treatment at a rotation speed of 10000-20000 rpm for 1-50 minutes;

[0029] The cell disruption and dispersion is as follows: the dispersion time is 1 to 5 minutes under the condition of power 100W;

[0030] The heating cross-linking curing is: reacting at a stirring speed of 200-500 rpm and 30-60° C. for 4-12 hours (thereby achieving interfacial polymerization of aldehyde groups and amine groups to solidify the carrier particles).

[0031] As a further improvement to the method for preparing ROS-responsive functionalized starch-based drug-loaded nano / microspheres of the present invention, the organic solvent in step 2) is at least any one of the following (i.e., a combination of one or more):

[0032] Salicylic acid, methyl salicylate, methyl jasmonate, methyl benzoate, ethylene dichloride, cyclohexane, hexane, cyclohexanone, methyl myristate, methyl hexadecanoate, and xylene.

[0033] As a further improvement to the method for preparing the ROS-responsive functionalized starch-based drug-loaded nano / microspheres of the present invention, the surfactant in step 2) is at least any one of the following (i.e., a combination of one or more):

[0034] Sodium dodecyl sulfate (SDS), sodium lignin sulfonate, sodium dodecylbenzene sulfonate, cocamidopropyl betaine, fatty alcohol polyoxyethylene ether series, Tween-80, alkyl polyglycoside APG-0810, alkylphenol polyoxyethylene ether, fatty alcohol polyoxyethylene ether, cetyltrimethylammonium bromide, silicone;

[0035] The fatty alcohol polyoxyethylene ether series includes fatty alcohol polyoxyethylene ether 23 (AEO-23).

[0036] As a further improvement to the preparation method of the ROS-responsive functionalized starch-based drug-loaded nano / microspheres of the present invention, the pesticide in step 2) is a fat-soluble pesticide;

[0037] Fat-soluble pesticides include fat-soluble fungicides and fat-soluble insecticides.

[0038] The fat-soluble fungicide is at least any one of the following: triadimefon, tebuconazole, propiconazole, difenoconazole, carbendazim, thiophanate-methyl, azoxystrobin, kresoxim-methyl, pyraclostrobin and the like;

[0039] The fat-soluble insecticide is at least any one of the following: avermectin, beta-cypermethrin, fipronil, chlorpyrifos, indoxacarb and other insecticides.

[0040] As a further improvement to the method for preparing ROS-responsive functionalized starch-based drug-loaded nano / microspheres of the present invention, the surfactant (preferably) in step 2) is a mixture of fatty alcohol polyoxyethylene ether 23 (AEO-23): sodium dodecyl sulfate (SDS) = 1:1 (w / w) weight ratio, and in the aqueous solution containing the surfactant, the concentrations of fatty alcohol polyoxyethylene ether 23 (AEO-23) and sodium dodecyl sulfate (SDS) are each 0.2 g / 100 ml.

[0041] Note: Relatively speaking, when the surfactants with the above preferred dosage and composition are used, the particle size of the prepared tebuconazole-loaded starch-based nanomicrospheres is the smallest.

[0042] The present invention also provides ROS-responsive functionalized starch-based drug-loaded nano / microspheres obtained by any of the above methods; the nano / microspheres have any of the following scales: nanoscale of 1 to 100 nm, submicron scale of greater than 100 to less than 1000 nm, or micron scale of 1 μm to 50 μm.

[0043] The present invention also provides the use of the above-mentioned ROS-responsive functionalized starch-based drug-loaded nano / microspheres (external protection and internal regulation):

[0044] It has the function of releasing pesticides in response to the presence of ROS (therefore, it can be used to prepare drug controlled release systems targeting high ROS environments, which can quickly respond to the environment and release drugs to increase the local concentration, achieving a rapid antibacterial effect);

[0045] It can activate the plant's own antioxidant defense regulatory function (therefore it can be used to prepare green agricultural carriers that simultaneously enhance drug efficacy and the plant's own defense capabilities).

[0046] In practice: The fungicide is delivered to the stem vascular tissues via foliar spraying and utilizing the transport properties of the carrier.

[0047] Specifically:

[0048] This invention relates to the preparation of starch-modified controlled-release drug-loaded nano / microcapsules and their application in disease control. The invention utilizes adjustable particle size of starch-based functional nano-delivery systems—drug-loaded nano / microcapsules—constructed via emulsion polymerization using amine-aldehyde condensation, and utilizes a controlled-release "ROS-responsive switch" to adjust the release rate of the drug-loaded system. This invention offers the advantages of simplicity, feasibility, ease of process scalability, and affordability.

[0049] The research of this invention is based on the ROS burst when the disease occurs as a regulatory factor. It is mainly based on the absorption characteristics of plant leaves. A starch-based nano / micro drug delivery system with polysaccharide-based ROS response function is rationally designed and prepared. The fungicide is delivered to the stem vascular tissue by spraying on the leaves and utilizing the transport characteristics of the carrier. An innovative design concept and application scheme of a composite intelligent nano-delivery system is proposed, aiming to simultaneously achieve targeted delivery of chemical pesticides (such as fungicides) and steady-state regulation of crop oxidative bursts caused by pathogens, and ultimately establish a prevention and control system for apple tree rot disease with "external prevention and internal regulation".

[0050] During the invention process, the present invention fully considered the following problems to be solved:

[0051] With regard to the existing patent literature, some are modified starch-based microcapsule drug delivery systems synthesized through multiple complex steps, and some are complex systems in which starch-based modified materials are only used as common excipients to prepare pesticide formulations. The particle sizes are all at the micron level, and the detailed controlled release or corresponding release function is unclear. No more ideal starch-based drug delivery controlled release nano / micro systems with ROS response and oxidation regulation functions have been reported, especially no preparation method for starch-based controlled release nano / micro systems modified with widely available, biodegradable carboxymethyl starch as the main material, which has both ROS response and oxidation regulation functions and controllable particle size.

[0052] The inventor's research group conducted in-depth research and, based on a comprehensive consideration of current research reports, found that by coupling a small molecule thioketal fragment (PDSE) with a ROS-sensitive site that can respond to release to the carboxyl group of carboxymethyl starch to enhance its hydrophobicity, and then using a multi-aldehyde cross-linker (TFPT), further amine aldehyde cross-linking can be achieved on the basis of interfacial polymerization to load pesticides and solidify the particles at the same time, aiming to simultaneously achieve targeted delivery of fungicides and steady-state regulation of crop oxidative bursts, and ultimately obtain an integrated prevention and control effect of "external prevention and internal regulation" for perennial fruit tree stem diseases.

[0053] Specifically, to improve the utilization rate of existing pesticides in practical applications, the present invention has developed a method for preparing starch-based sustained-release nano / microspheres with simple processes, ingenious design, functionalization, and controllable particle size. By selecting a suitable functional small molecule (PDSE) capable of ROS-responsive release and attaching it to the starch moiety, the system is then crosslinked with a suitable organic solvent, acting as a plant defense-related signaling molecule to regulate plant growth or defense, while also providing an oil phase for interfacial polymerization, enabling the construction of nanostructured systems and pesticide encapsulation. By regulating the preparation method and conditions of the functional carrier material (such as the type of surfactant and the amount of loaded pesticide), the present invention not only enables the regulation of drug release rate from drug-loaded nano / microsphere encapsulation, but also possesses the special features of ROS-responsive release and antioxidant properties. For perennial trees (such as apple trees), the system not only offers the rapid efficacy of external spraying for disease control, but also has internal conditioning effects. Furthermore, the system exhibits oxidative stress response properties, enabling rapid drug release in the oxidative burst environment within the plant body during the initial stages of pathogen infection, blocking pathogen spread and improving the effectiveness of control. This technology offers significant technical advantages. In addition, the drug-loaded material of the present invention has a responsive function, is highly safe for crops, can be gradually decomposed in the environment, and can effectively avoid environmental pollution problems caused by microplastic residues.

[0054] This invention is based on the ROS burst during the occurrence of important forest diseases, such as apple rot, as a regulatory factor. It innovatively and rationally designs a composite intelligent nano-delivery system and studies its application scheme. It can simultaneously achieve targeted delivery of existing commercial agents and steady-state regulation of crop oxidative bursts, and ultimately establish a prevention and control system for forest diseases with "external prevention and internal regulation".

[0055] Compared with the prior art, the present invention has the following technical advantages:

[0056] 1. The present invention uses carboxymethyl starch, which is widely available, environmentally friendly, biocompatible and naturally degradable, as raw material. Combining structural advantages with bioaffinity, a simple, mild chemical modification method is used to construct a pesticide delivery nano / micro system with controllable particle size and ROS response function.

[0057] 2. The present invention achieves controllable particle size of starch-based drug-loaded nano / microspheres by regulating the type and amount of added surfactants, so that the release characteristics and duration of the agent can be precisely targeted according to the needs of different crops and diseases.

[0058] 3. The nanoscale drug-loaded balls described in the present invention have good directional delivery capabilities in the phloem of plants, which can effectively achieve precise prevention and control of hidden or vascular diseases and improve the utilization efficiency of drugs.

[0059] 4. The nano / micron-scale drug-loaded balls described in the present invention have a sustained-release function, which can achieve the slow release of pesticides after application, thereby extending the duration of drug efficacy and reducing the number of applications.

[0060] 5. The starch-based drug-loaded nano / microspheres constructed in the present invention have the characteristics of responsiveness to oxidative stress (ROS), and can quickly release drugs in the oxidative burst environment in the plant body at the early stage of pathogen infection, thereby blocking the spread of pathogens and improving the timeliness of prevention and control.

[0061] 6. The nano / microsphere system of the present invention can synergistically deliver plant defense-related signal molecules, help activate the plant's own antioxidant system, regulate the body's oxidative stress response, and reduce the risk of programmed death of healthy tissues caused by excessive defense.

[0062] 7. The starch-based drug-loaded nano / microspheres involved in the present invention are recommended to be applied by foliar spraying or stem injection as the main method of application, avoiding the damage to plant tissue caused by physical scraping of diseased tissue in existing prevention and control methods, reducing the risk of disease spread, and having higher practicality and safety.

[0063] In summary, the present invention uses carboxymethyl starch, which is widely available and biodegradable, as the main material, and has both ROS response and oxidation regulation functions and controllable particle size. The method uses a small molecule fragment of ketal thiol (PDSE) with a ROS sensitive site as the hydrophobic end of the modified carboxymethyl starch, which is coupled to its hydrophilic end carboxyl group, and then uses a customized multi-aldehyde cross-linking agent, preferably a suitable plant defense signal molecule (such as salicylic acid, etc.) as an organic solvent to dissolve the pesticide as the oil phase, and further realizes amine aldehyde cross-linking on the basis of interfacial polymerization to This invention aims to simultaneously achieve targeted delivery of pesticides and homeostatic regulation of crop oxidative stress, ultimately achieving an integrated "external defense and internal regulation" control of perennial fruit tree stem diseases. This strategy combines external defense (resistance to external pathogen invasion) and internal regulation (regulation of oxidative stress within the plant). The invention innovatively constructs a multiphase nano-delivery system capable of simultaneously loading a fungicide (such as tebuconazole) and a plant defense signaling molecule (such as methyl salicylate) to enhance the integrated control of perennial forest pests and diseases. Results show that the drug delivery system not only possesses the essential properties of conventional nano / micro drug delivery systems, such as improved leaf retention and dispersion, enhanced absorption and transduction, and better targeted delivery, but also rapidly responds to ROS bursts by releasing the fungicide while maintaining redox balance by scavenging excess ROS. This self-regulatory mechanism mitigates damage to healthy tissues caused by high ROS levels and slows drug release by reducing ambient ROS concentration, thereby extending the drug's duration of action. The technology of the present invention provides a new idea for the green prevention and control of perennial forest diseases and insect pests, in order to achieve the "external prevention and internal adjustment" prevention and control of forest diseases and insect pests, such as apple tree rot, from the inside out. This strategy is expected to have the dual advantages of simple operation and breaking through the limitations of existing prevention and control technologies. It provides a new strategy for the development of composite functional nanotechnology, which is of great significance to the reduction and efficiency of pesticides and green plant protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 The H NMR spectrum ( 1 H NMR);

[0065] Figure 1 middle:

[0066] A is the H NMR spectrum of the ROS-responsive molecule used in Example 1, 2,2'-(propane-2,2-diylbis(sulfonamide))diethylamine (PDSE); its structural formula is: ;

[0067] B is the H NMR spectrum of the crosslinker molecule, 2, 4, 6-tris(4-formylphenoxy)-1, 3, 5-triazine (TFPT); its structural formula is: ;

[0068] C is the H NMR spectrum of carboxymethyl starch and PDSE-modified (covalently modified) carboxymethyl starch, and its structural formula is: .

[0069] Figure 2 is the Fourier transform infrared spectrum;

[0070] Figure 2 middle:

[0071] a is a Fourier transform infrared spectra of carboxymethyl starch, PDSE-modified carboxymethyl starch and blank starch-based nanoparticles / microspheres (i.e., ROS-responsive functionalized starch-based drug-loaded nanoparticles / microspheres) in Example 1;

[0072] b is the Fourier transform infrared spectra of tebuconazole, blank starch base nanoparticles / microspheres, and tebuconazole starch base nanoparticles / microspheres in Example 3.

[0073] Figure 3 This is a curve diagram of the release rate of the starch-based nano / microspheres loaded with tebuconazole prepared in Example 3 under conditions of different hydrogen peroxide concentrations.

[0074] Figure 4 This is a visualization of the delivery and distribution of tebuconazole-loaded starch nanoparticles / microspheres prepared in Example 3 in apple seedlings (laser confocal image);

[0075] Figure 4 middle:

[0076] a is a healthy seedling, and the scales from top to bottom are: 500 μm, 100 μm, 200 μm, and 200 μm;

[0077] b is the ring-cut seedling; the scales from top to bottom are: 500 μm, 200 μm, 300 μm, 500 μm.

[0078] Figure 5 This is a graph showing the control effect of tebuconazole-loaded starch-based nanoparticles / microspheres prepared in Example 3 on apple rot;

[0079] Figure 5 middle:

[0080] A is the prevention effect diagram;

[0081] B is the ROS (hydrogen peroxide) content in the diseased tissue of apple seedlings.

[0082] Figure 6 This is a data graph showing changes in ROS content and antioxidant enzyme activity in diseased tissues of apple seedlings after treatment with starch-based nanoparticles / microspheres loaded with tebuconazole in Example 3;

[0083] Figure 6middle:

[0084] A is the ROS (hydrogen peroxide) content in the diseased tissue of apple seedlings;

[0085] B is CAT enzyme;

[0086] C is POD enzyme;

[0087] D is SOD enzyme.

[0088] Figure 7 This is a data graph showing the effect of blank starch microspheres / microspheres on the activities of defense-related enzymes in apple seedlings in Example 1;

[0089] Figure 7 middle:

[0090] a is PPO enzyme;

[0091] b is PAL enzyme;

[0092] c is POD enzyme. DETAILED DESCRIPTION

[0093] The present invention will be further described below with reference to specific embodiments, but the protection scope of the present invention is not limited thereto.

[0094] The nuclear magnetic resonance hydrogen spectrometer used in the examples was an AVANCE III 400 h z; the transmission electron microscope was an HT-7700 field emission transmission electron microscope (Hitachi, Japan); the thermal field emission scanning electron microscope was a Zeiss G300; the liquid chromatograph was a Shimadzu LC-10AT; and the particle size analyzer was a Nano-ZS90 laser particle size analyzer.

[0095] 2, 2'-(Propane-2, 2-diylbis(sulfonamide))diethylamine (PDSE), a ROS-responsive switch, and 2, 4, 6-tris(4-formylphenoxy)-1, 3, 5-triazine (TFPT), a cross-linking agent, are both known compounds.

[0096] 2,2'-(Propane-2,2-diylbis(sulfonamide))diethylamine (PDSE) can be prepared, for example, by the following method:

[0097] 15 g of cysteamine hydrochloride was dissolved in 8 ml of 37.5% hydrochloric acid at 30°C with stirring. A mixture of 25 ml of acetone and 10 ml of dichloromethane was then added dropwise to the solution while stirring in an ice bath at 0°C. The reaction was continued with stirring for 6–12 hours. After the reaction, the white solid precipitate was collected by filtration and then completely dissolved in a near-saturated sodium hydroxide solution. The product was extracted by repeated additions of dichloromethane. Finally, anhydrous sodium sulfate was added to remove excess water, and the dichloromethane was removed by rotary evaporation under reduced pressure to obtain the final product, 2,2'-(propane-2,2-diylbis(sulfonamide))diethylamine (PDSE).

[0098] 2, 4, 6-tris(4-formylphenoxy)-1, 3, 5-triazine (TFPT) can be prepared, for example, by the following method:

[0099] Dissolve 15 g of 4-hydroxybenzaldehyde and 6 g of sodium hydroxide in 60 ml of a 1:1, v / v, acetone-water mixture. Dissolve 10 g of cyanuric chloride in an appropriate amount of acetone (just enough to dissolve the cyanuric chloride). Add the resulting cyanuric chloride solution dropwise to the 4-hydroxybenzaldehyde and sodium hydroxide mixture while stirring in an ice bath. After the addition is complete, heat the reaction system to 50°C and stir continuously for 4-12 hours, resulting in the formation of a white precipitate. Disperse the precipitate in water with mechanical stirring, wash thoroughly three times with deionized water, and then recrystallize the product from ethanol to obtain TFPT.

[0100] Carboxymethyl starch (CMS): available from Maclean, reagent grade sodium carboxymethyl starch, product number: C807411-500g, CAS number: 9063-38-1, viscosity 600-3000 mPa·s.

[0101] Example 1. Preparation of ROS-responsive starch-based blank nano / microspheres

[0102] (1) ROS-responsive starch-based wall material PDSE-CMS:

[0103] 1.5 g of carboxymethyl starch (CMS) was dispersed in 50 ml of phosphate buffer (pH 6.0, 0.01 M). 600 mg of EDC and 500 mg of NHS were then added to the solution and stirred at 50°C for 1 hour to activate the carboxyl groups on the CMS molecular chains. Subsequently, 5 g of PDSE was added to the activated solution and stirred at 50°C for 24 hours, at which point the solution became transparent. After the reaction, the resulting liquid product was placed in a 5000 Da dialysis bag and dialyzed with deionized water until the pH of the external medium approached neutral (dialysis time was approximately 48 hours). Finally, the dialyzed solution (in the dialysis bag) was freeze-dried (from 0 to -20°C to constant weight) to obtain a pale yellow powder product: PDSE-covalently modified carboxymethyl starch (PDSE-CMS), i.e., the ROS-responsive starch-based wall material PDSE-CMS.

[0104] (2) Preparation of ROS-responsive starch-based blank nano / microspheres

[0105] 2.1) Dissolve 100 mg of PDSE-CMS in 100 ml of an aqueous solution containing a surfactant as the aqueous phase; the surfactant is composed of fatty alcohol polyoxyethylene ether 23 (AEO-23) and sodium dodecyl sulfate (SDS); the mass ratio of AEO-23:SDS is 1:1, and the concentrations of AEO-23 and SDS in the aqueous solution containing the surfactant are both 0.2 g / 100 ml;

[0106] 2.2) Take 1 ml of methyl salicylate as the organic phase and dissolve 25 mg of the cross-linking agent (TFPT) in it;

[0107] 2.3) In an ice bath, the aqueous phase and the oil phase were mixed and initially emulsified at a shear rate of 10,000 rpm for 5 minutes using a high-shear emulsifier. The emulsion particle size was then further refined using a cell disruptor (cell disruptor power of 100 W, dispersion time of 1 minute). Subsequently, the emulsion (after homogenization) was transferred to a magnetic stirrer and stirred at 200 rpm for 3 hours to achieve interfacial polymerization of the aldehyde and amine groups to obtain solidified carrier particles (the resulting suspension was a suspension of ROS-responsive starch-based blank nano / microspheres). Measurements using a BT-9300S laser particle size analyzer showed that the average particle size of the nanoparticles obtained by this method was approximately 80.0 nm.

[0108] Example 2. Preparation of ROS-responsive starch-based fluorescent nano / microspheres

[0109] The ROS-responsive starch-based wall material PDSE-CMS and cross-linking agent prepared in Example 1 were used, with the following changes relative to Example 1:

[0110] The use of "Fluorescein-Nile Red" has been added;

[0111] That is, the step 2.2) was modified to "dissolve 25 mg of fluorescein-Nile red and 25 mg of cross-linking agent (TFPT)" and the rest was the same as in Example 1.

[0112] According to the measurement results of the laser particle size analyzer BT-9300S, the average particle size of the nanoparticles obtained by this method is about 90.0 nm.

[0113] Example 3. Preparation of ROS-responsive starch-based drug-loaded nano / microspheres

[0114] The ROS-responsive starch-based wall material PDSE-CMS and cross-linking agent prepared in Example 1 were used, with the following changes relative to Example 1:

[0115] Increased use of "pesticides";

[0116] That is, the step 2.2) was modified into “dissolving 25 mg of the cross-linking agent (TFPT)” and the rest was the same as in Example 1.

[0117] According to the measurement results of the laser particle size analyzer BT-9300S, the average particle size of the nanoparticles obtained by this method is about 97.8 nm.

[0118] Example 4. Preparation of ROS-responsive starch-based drug-loaded nano / microspheres

[0119] The ROS-responsive starch-based wall material PDSE-CMS and cross-linking agent prepared in Example 1 were used, with the following changes relative to Example 1:

[0120] Eliminate the use of surfactant in step 2.1), that is, replace the aqueous solution containing surfactant with water, and keep the amount unchanged at 100 ml;

[0121] In step 2.2), the phrase "Take 1 ml of methyl salicylate as the organic phase and dissolve 25 mg of the cross-linking agent (TFPT) therein" was changed to "Take 1 ml of methyl salicylate as the organic phase and dissolve 25 mg of tebuconazole and 25 mg of the cross-linking agent (TFPT) therein";

[0122] Change "Stir and react for 3 hours" in step 2.3) to "Stir and react for 6 hours";

[0123] The rest is the same as Example 1.

[0124] According to the measurement results of the laser particle size analyzer BT-9300S, the average particle size of the nanoparticles obtained by this method is about 3877.85 nm.

[0125] Example 5. Preparation of ROS-responsive starch-based drug-loaded nano / microspheres

[0126] The ROS-responsive starch-based wall material PDSE-CMS and cross-linking agent prepared in Example 1 were used, with the following changes relative to Example 1:

[0127] Change the step 2.2) from "Take 1 ml of methyl salicylate as the organic phase and dissolve 25 mg of the cross-linking agent (TFPT) in it" to "Take 2 ml of methyl salicylate as the organic phase and dissolve 125 mg of tebuconazole and 25 mg of the cross-linking agent (TFPT)";

[0128] Change "Stir and react for 3 hours" in step 2.3) to "Stir and react for 6 hours";

[0129] The rest is the same as Example 1.

[0130] According to the measurement results of the laser particle size analyzer BT-9300S, the average particle size of the nanoparticles obtained by this method is about 246.6 nm.

[0131] Example 6. Preparation of ROS-responsive starch-based drug-loaded nano / microspheres

[0132] The ROS-responsive starch-based wall material PDSE-CMS and cross-linking agent prepared in Example 1 were used, with the following changes relative to Example 1:

[0133] Change the step 2.2) from "Take 1 ml of methyl salicylate as the organic phase and dissolve 25 mg of the cross-linking agent (TFPT) in it" to "Take 1 ml of methyl jasmonate as the organic phase and dissolve 25 mg of tebuconazole and 25 mg of the cross-linking agent (TFPT)";

[0134] Change "Stir and react for 3 hours" in step 2.3) to "Stir and react for 6 hours";

[0135] The rest is the same as Example 1.

[0136] According to the measurement results of the laser particle size analyzer BT-9300S, the average particle size of the nanoparticles obtained by this method is about 156.6 nm.

[0137] Example 7. Preparation of ROS-responsive starch-based drug-loaded nano / microspheres

[0138] The ROS-responsive starch-based wall material PDSE-CMS and cross-linking agent prepared in Example 1 were used, with the following changes relative to Example 1:

[0139] Change the step 2.2) from "Take 1 ml of methyl salicylate as the organic phase and dissolve 25 mg of the cross-linking agent (TFPT) in it" to "Take 2 ml of methyl salicylate as the organic phase and dissolve 25 mg of the insecticide indoxacarb and 25 mg of the cross-linking agent (TFPT)";

[0140] The rest is the same as Example 1.

[0141] According to the measurement results of the laser particle size analyzer, the average particle size of the nanoparticles obtained by this method is 107.0 nm.

[0142] According to conventional scanning electron microscopy, the microspheres of the present invention are regular spherical in shape and have a relatively smooth surface.

[0143] Structural characterization of prepared materials:

[0144] In order to demonstrate the successful preparation of ROS-responsive molecules, crosslinkers, and thioketal-based starch, the ROS-responsive molecule in Example 1, 2, 2'-(propane-2, 2-diylbis(sulfonamide))diethylamine (PDSE), and the crosslinker molecule, 2, 4, 6-tris(4-formylphenoxy)-1, 3, 5-triazine (TFPT), were subjected to H NMR spectroscopy. The results are shown in Figure 2. Figure 1 As shown in A and B.

[0145] The characteristic peaks of ROS response molecules were observed in the NMR spectrum ( δ = -CH3 groups at 1.51 ppm, δ = 2.64 and 2.80 ppm), and the aldehyde group and H on the benzene ring skeleton also appeared in the NMR spectrum of the cross-linker ( δ = -CHO group at 10.0, δ = 7.52 and 7.98 at the -CH group), confirming its successful synthesis. In addition, ketalthioate starch (ROS-responsive starch-based wall material PDSE-CMS) 1 The H NMR spectrum shows the characteristic chemical shift peaks of ROS response molecules ( Figure 1 C), demonstrating the successful preparation of thioketal-based starch.

[0146] In order to prove the synthesis of starch-based nanoparticles and the successful encapsulation of tebuconazole, the starch-based nanoparticles loaded with tebuconazole prepared in Example 3 were characterized by Fourier transform infrared spectroscopy. Figure 2 As shown in the infrared spectrum of carboxymethyl starch, 3800 ~ 3000 cm -1 The broad absorption band in the range of 1610 cm corresponds to the stretching vibration of hydroxyl (-OH) -1The absorption band at 1580 cm can be attributed to the overlap of C=O stretching vibration of -COONa in carboxymethyl starch. In addition, in the infrared spectrum of PDSE-modified carboxymethyl starch, the absorption band at 1580 cm -1 NH bending vibration appeared at 1650 cm-1, and these characteristic peaks together confirmed the successful reaction between PDSE and CMS. In the infrared spectrum of starch-based nano / microspheres without drug loading, the C=N stretching vibration of the crosslinker overlaps with the C=O stretching vibration of the carboxyl group, resulting in -1 The absorption peak at 1230 cm becomes more obvious. -1 The absorption peak at 1750 cm corresponds to the CO stretching vibration in the ether bond connected to the benzene ring. These results further confirm the successful cross-linking of the cross-linker with PDSE-modified carboxymethyl starch. -1 and 1910cm -1 The two characteristic absorption peaks at 1510 cm-1 belong to the overtone vibration of benzene derivatives, while the -1 The stretching vibration peak band of the triazole ring was observed, confirming that the fungicide tebuconazole was successfully loaded into the carrier material.

[0147] The following experiment was conducted on the ROS-responsive starch-based tebuconazole-loaded nano / microspheres (abbreviated as Teb@PT-CMS, where Teb is the abbreviation of tebuconazole) with an average particle size of 97.8 nm prepared in Example 3.

[0148] Experiment 1: Drug release test of ROS-responsive starch-based drug-loaded nano / microspheres:

[0149] The rapid burst and accumulation of ROS is one of the most rapid defense responses of host plants against pathogen invasion. Based on this theory, the present invention has developed a ROS-responsive nanodelivery system to regulate the release rate of loaded drugs according to the fluctuation of ROS levels in plants.

[0150] This function was verified by in vitro drug release experiments simulating multiple ROS stimulations. Given that H2O2 is one of the main and most stable ROS in plants, the effect of H2O2 concentration changes on drug release was mainly tested.

[0151] Using 0.5% Tween-80 aqueous solution as the release medium, the effect of different H2O2 concentrations on the release rate of tebuconazole from starch-based sodium / microspheres loaded with tebuconazole was analyzed by in vitro dialysis experiments.

[0152] Specific operation: 2 ml of Teb@PT-CMS (nanoparticles prepared in Example 3) suspension was added to dialysis bags containing different concentrations of H2O2, where the final concentrations of H2O2 were 0, 50, and 50 μM, respectively, and then placed in 100 ml of release medium (0.5% Tween-80 aqueous solution). The release experiment was carried out in a constant temperature shaker at 25°C and a speed of 100 rpm. After 1 hour of the experiment, H2O2 was added to one of the dialysis bags containing 50 μM H2O2 to reach a final concentration of 100 μM (i.e. Figure 3 The release rate was analyzed by measuring the effect of varying H2O2 concentrations (50 μM x 2). The external buffer medium was collected at specific intervals (1, 2, 4, 6, 8, 12, 24 hours, etc.) and replaced with an equal volume of release medium. The collected drug-containing medium was filtered through a 0.45 μm organic filter membrane and assayed for tebuconazole content by HPLC. The cumulative percentage was calculated using the following formula. The cumulative percentage of tebuconazole released from ROS-responsive starch-loaded drug-loaded nano / microspheres was calculated using the following formula.

[0153]

[0154] Where, t represents the cumulative release time at each sampling (h); M t represents the mass of tebuconazole released from the nanoparticles at the sampling time point; M 0 represents the total mass of tebuconazole in the initially added nanoparticles.

[0155] The results are as follows Figure 3 As shown. There are significant differences in the release kinetics of tebuconazole-loaded starch-based nanoparticles (drug-loaded microspheres obtained in Example 3) under different H2O2 treatment conditions. Compared with the blank control group without H2O2 addition, the group treated with an initial addition of 50 μM hourly H2O2 at a final concentration of 50 μM hourly gradually showed differences in the cumulative drug release rate after 24 hours of release. In addition, compared with the group treated with a single addition of H2O2 (50 μM×1), the group treated with a second additional addition of 50 μM hourly H2O2 (100 μM cumulative) at 24 hours ( Figure 3The release rate was faster in the 0 μM, 50 μM × 1, and 100 μM cumulative concentration (i.e., 50 μM × 2 treatment groups). After 60 hours of release, the cumulative release rates of tebuconazole from the starch-based nanoparticles / microspheres reached 70.0% ± 1.3%, 83.3% ± 0.3%, and 97.5% ± 0.9%, respectively, for the 0 μM, 50 μM × 1, and 100 μM cumulative concentration (i.e., 50 μM × 2 treatment groups). After 123 hours of release, the release curve in the 100 μM cumulative concentration treatment group flattened out, while that in the blank control group did not flatten out until 264 hours. These data strongly confirm that the addition of H2O2 significantly accelerates the release rate of tebuconazole, and that the ROS-responsive release rate of tebuconazole from the starch-based nanoparticles / microspheres loaded with tebuconazole exhibits a dose-dependent effect on H2O2.

[0156] Experiment 2: Absorption and transport of ROS-responsive starch-based drug-loaded nano / microspheres in apple seedlings

[0157] Fluorescence tracer analysis was used to analyze the absorption and transport pathways of fluorescein- and tebuconazole-loaded starch-based nanoparticles / microspheres prepared in Examples 2 and 3 by apple seedling leaves. Briefly, a suspension of starch-based nanoparticles / microspheres labeled with Nile Red was evenly applied to each leaf surface. To ensure that the agent was absorbed and effective only through the leaves, the seedlings were placed horizontally during spraying, and the stems and roots were wrapped with plastic film to isolate the solution. After air-drying, the seedlings were placed in a greenhouse for further cultivation. The seedlings were then placed in a greenhouse for further cultivation. After 24 hours of treatment, the apple seedlings were divided into three sections: roots, stems, and leaves, and slides were prepared. The distribution of excitation fluorescence from the starch-based nanoparticles / microsphere suspension in different tissues was then observed using a laser confocal microscope (FV3000). The Nile Red fluorescence signal was detected using an excitation wavelength of 528 nm and a detection wavelength of 576 nm. To further verify the long-distance transport pathway of nanoparticles, the middle section of the stem of apple seedlings was ring-cut (about 2.0 cm wide) to block the phloem transport pathway of organic matter. Figure 4 Middle: The treated leaf refers to the drug-treated leaf above the ring-cut stem (i.e., the leaf directly coated with the nanoparticles loaded with Nile Red), and the lower treated leaf refers to the non-drug-treated leaf below the ring-cut stem. This is the default common sense in the industry. After 48 hours of blocking, the same fluorescence treatment method as above was used to observe the distribution of nanoparticles in the roots, stems and leaves of the ring-cut samples. Finally, the fluorescence distribution difference between the ring-cut treated and untreated samples was compared to verify the results. Figure 4As shown. Laser confocal microscopy was used to observe the fluorescence distribution of apple seedling leaves treated with starch-based nanoparticles / microspheres loaded with Nile red for 24 hours. The results showed that red fluorescence signals of Nile red were detected in the lower leaves, stems and roots of healthy apple seedlings, indicating that the carrier nanoparticles / microspheres were successfully transported downward in the apple seedlings. It is worth noting that the fluorescence signal was not only distributed in the vascular tissue, but also significant punctate fluorescence was observed in the outer cortex of the phloem of the stems and roots. This phenomenon can be attributed to the lipophilicity of Nile red, which binds to it in a lipid-rich environment and emits strong fluorescence. To further confirm that the long-distance transport of nanoparticles is mediated by the phloem, the main stem of the apple tree was ring-cut with a width of 2 cm. This treatment is often used to block the basal transport of nutrients to improve the fruit set rate and fruit quality at the upper end. Laser confocal microscopy images showed that when the phloem transport pathway was blocked, the Nile Red fluorescence signal in all organs at the lower end of the leaves treated with starch-based nanoparticles / microspheres loaded with Nile Red completely disappeared, indirectly verifying the conclusion that the carrier nanoparticles / microspheres rely on phloem for long-distance transport.

[0158] Experiment 3: Control of apple rot with ROS-responsive starch-based drug-loaded nano / microspheres

[0159] The inoculation process was performed using a method called "applying a bacterial cake" after applying the pesticide. A wound was first created on the stem using an inoculating knife. A 5mm diameter bacterial cake, grown for five days on potato dextrose agar, was then applied to the wound, gradually inducing disease in the apple seedlings. The efficacy of the starch-based nanoparticles / microspheres loaded with tebuconazole, prepared in Example 3, against apple rot in potted plants was evaluated. An aqueous suspension of a commercial tebuconazole suspension concentrate was prepared (with the same effective concentration as in Example 3). The negative control consisted of healthy apple seedlings treated with sterile water and not inoculated. The positive control consisted of diseased apples treated with sterile water but inoculated with the pathogen. The solution was then evenly sprayed onto the leaves of the apple seedlings at a rate of 20 ml per seedling. To ensure that the solution was absorbed only through the leaves, the seedlings were placed horizontally during spraying, and the stems and roots were wrapped with plastic film to isolate the solution (this is standard practice). After air-drying, the seedlings were placed in a greenhouse for further cultivation. After 7 and 14 days of incubation, the main stems of the experimental seedlings were cut into 15-cm-long branches (measured across the entire plant, starting at the intersection of the branch with the last leaf and the main stem, and proceeding every 15 cm). The branches were rinsed with running water to remove surface dust, then disinfected with 0.06% sodium hypochlorite and 75% ethanol, followed by a thorough rinse with sterile water. The ends of the branches were sealed with paraffin wax for preservation. All branches were then incubated for 7 days in an artificial incubator at 25°C, 90% relative humidity, and a 16-hour:8-hour light-dark cycle. After 7 days, the longitudinal length of the lesions on the branches was measured, and the protection efficiency was calculated according to the following formula. Finally, bark from all branches was collected and stored at -80°C for subsequent analysis.

[0160]

[0161] Figure 5 The results show that the effects of Tebuconazole starch-based microsphere suspension and Tebuconazole commercial suspension in water on stems were observed 7 or 14 days after foliar application. Valsa mali 03-8Infected lesions. Figure 5 In the positive control group, Valsa maliAfter inoculation with the 03-8 fungus cake, the bark developed varying degrees of ulcerative necrosis. Scraping the bark revealed a dark brown discoloration of the xylem. However, the longitudinal diameter of the canker lesions decreased in the groups sprayed with both the aqueous suspension of tebuconazole starch-based nanospheres and the commercial tebuconazole suspension. Furthermore, the xylem discoloration was minimal in the tebuconazole-loaded starch-based nanospheres, indicating effective inhibition of the lateral spread of the pathogen. Seven and fourteen days after application, the protective efficacy of the tebuconazole-loaded starch-based nanospheres was 54.0% and 22.4%, respectively, representing 2.4 and 2.1 times that of the tebuconazole-loaded suspension. The ability of the tebuconazole-loaded starch-based nanospheres to modulate oxidative stress was systematically evaluated by quantitatively measuring H₂O₂ concentrations in bark tissue infected with the pathogen. Results showed that H₂O₂ concentrations in the bark treated with tebuconazole starch-based nanospheres were significantly lower than those in the blank inoculation group and the tebuconazole suspension group.

[0162] Experiment 4: Effect of ROS-responsive starch-based drug-loaded nano / microspheres on ROS content and antioxidant enzyme activity in diseased tissues of apple seedlings infected with apple rot:

[0163] For each of the different apple seedlings treated in Experiment 3, 100 mg of bark tissue was collected and thoroughly ground in liquid nitrogen. The ground bark tissue was then mixed with 1 mL of an extract containing 0.25 mL of trichloroacetic acid, 0.5 mL of potassium iodide, and 0.25 mL of potassium dihydrogen phosphate buffer (10 mM, pH 6.5). The mixture was homogenized in an ice bath for 10 minutes and then centrifuged at 12,000 rpm and 4°C for 15 minutes. The supernatant was collected and incubated in the dark at 25°C for 15 minutes. The absorbance of the supernatant was then measured at 390 nm using a UV spectrophotometer. Additionally, standard H₂O₂ solutions with concentrations ranging from 0.625 to 500 μM were prepared. The absorbance was measured, and a scatter plot of the absorbance versus concentration was plotted. A linear regression equation was fitted to generate a standard H₂O₂ curve to calculate the H₂O₂ concentration in the samples.

[0164] For antioxidant enzyme activity testing, apple seedlings were pretreated using the inoculation-before-drug application method (see Experiment 3): The stems of the apple seedlings were evenly marked into 15-cm-long segments. The stems were then inoculated using a live inoculation method, using a smear-based inoculation method. A 5-mm-diameter wound was artificially created on the stems using an inoculation knife. A 5-day-old smear of smeared ... Bark samples collected during the aforementioned experiments were analyzed using superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) enzyme activity assay kits (Solaibao, product numbers: BC5160, BC0095, and BC0205, Beijing Solaibao Technology Co., Ltd.) according to the kit instructions.

[0165] The results are as follows Figure 6 As shown. Valsa mali In the 03-8 infection group (positive control), the H2O2 concentration first increased and then decreased with the treatment time. From the changes in antioxidant enzyme activity, pathogen infection mainly stimulates the increase of POD enzyme activity. POD can indirectly produce highly destructive hydroxyl radicals (·OH). At the same time, pathogens inhibit SOD activity, that is, inhibit the production of superoxide anions (O2 - ) into H2O2. Therefore, compared with healthy apple plants (negative control), the H2O2 level in the infected group was lower, but it also means that there may be higher O2 - and ·OH accumulation, damaging healthy stem tissue cells. In contrast, H2O2 concentrations in the tebuconazole starch base / microsphere treatment group were similar to those in the infected group on days 1 and 3, but decreased significantly on day 7. Analysis of antioxidant enzyme activity trends showed that application of tebuconazole starch base / microspheres containing methyl salicylate significantly enhanced CAT activity, an enzyme that directly catalyzes the hydrolysis of H2O2 without generating harmful byproducts. However, POD and SOD activities remained unchanged.

[0166] Experiment 5: Biosafety Evaluation of Blank Starch Kina / Microspheres

[0167] To evaluate the safety of the carrier material for the normal physiological processes of apple seedlings, a blank starch-based nanoparticle / microsphere carrier suspension prepared in Example 1 was evenly sprayed onto the entire plant (until liquid dripped). Healthy apple seedlings treated with the same spraying treatment using water served as a blank control. Leaf samples were collected 1, 3, and 7 days after treatment, and the activities of defense enzymes in these leaf samples were measured using phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO), and peroxidase (POD) enzyme activity assay kits (Solaibao, Product Nos. BC0215, BC0195, and BC0095, Beijing Solaibao Technology Co., Ltd.). Specific procedures were performed according to the kit instructions.

[0168] The results are as follows Figure 7 As shown, after application of a blank starch-based microsphere suspension, the activities of PAL and PPO were not significantly different from those of healthy plants, while POD enzyme activity was significantly enhanced one day after application. Therefore, the blank starch-based microsphere carrier is not recognized as a pest by apple plants, triggering typical defense responses such as lignin accumulation, cell wall thickening, and phenolic oxidation. The upregulation of POD enzymes, which are associated with antioxidant processes, may be attributed to the methyl salicylate in PT-CMS. Studies have shown that methyl salicylate and methyl jasmonate are commonly used to enhance the antioxidant system of fruit, thereby extending its storage life.

[0169] Comparative Example 1: Comparative drug release test of conventional starch-based drug-loaded nano / microspheres (verifying that they have no ROS-responsive release performance)

[0170] Compared with Example 3, "PDSE" was replaced with "ethylenediamine", and the amount used remained unchanged at 5 g; the rest was the same as Example 3.

[0171] Note: Ethylenediamine is a common reagent and has no ROS response function.

[0172] According to the drug release test method of Experiment 1 above, the ROS-responsive release results of ethylenediamine starch-based tebuconazole sodium / microspheres are shown in Table 1 below.

[0173] Table 1

[0174]

[0175] The difference in drug release was not significant, indicating that the ethylenediamine-modified starch-based nano-microspheres did not have the performance of ROS-responsive drug release.

[0176] Comparative Example 2: No crosslinking agent

[0177] Compared with Example 3: the use of “25 mg of cross-linking agent (TFPT)” is omitted; the rest is the same as Example 3.

[0178] The resulting emulsion was nearly transparent, and no spherical nanoparticles were visible under a scanning electron microscope. Without a crosslinker, the oil-water emulsion containing the starch-based material and the drug could not be cured and crosslinked to form spherical nano / microsphere shell structures.

[0179] Comparative Example 3: Ordinary solvents used as oil phase to dissolve pesticides

[0180] With respect to Example 3: "1 ml of methyl salicylate" was replaced with "1 ml of dichloromethane" as the organic phase; the rest was the same as Example 3.

[0181] The results of the experiment 4 were similar to those of the commercial suspension concentrate. Valsa mali In the 03-8 infection group, H2O2 concentrations gradually decreased over time. Analysis of the changing trends in antioxidant enzyme activity showed no significant changes in CAT activity after application of tebuconazole starch base sodium / microspheres containing dichloromethane, suggesting that the use of a common solvent as the oil phase, without methyl salicylate, had a greater impact on CAT activity.

[0182] Example 8. Preparation of ROS-responsive starch-based drug-loaded nano / microspheres (single surfactant)

[0183] Compared to Example 3, the surfactants were replaced with single adjuvants: cetyltrimethylammonium bromide (CTAB), cocamidopropyl betaine (CAB-35), alkyl glycoside APG-0810 (APG-0810), fatty alcohol polyoxyethylene ether O-20 (Peregal O-20), sodium lignin sulfonate (SL), etc., as shown in Table 2 below. The concentration of each in the aqueous solution containing the surfactants was 0.4 g / 100 ml. All other procedures were the same as in Example 3.

[0184] The particle size and physical appearance of a series of starch-based tebuconazole sodium / microspheres obtained in Example 8 are shown in Table 2 below:

[0185] Table 2

[0186]

[0187] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. Preparation method of ROS-responsive functionalized starch-based drug-loaded nano / microspheres, characterized in that The following steps are involved: 1) Prepare ROS-responsive starch-based wall material PDSE-CMS using carboxymethyl starch: Carboxymethyl starch was added to a phosphate buffer solution, followed by an activator. The mixture was stirred at 40 ± 10°C for 1–5 hours. PDSE, a ROS-responsive switch, was then added and stirred at 20–80°C for 24 ± 1 hour. The ROS-responsive starch-based wall material, PDSE-CMS, was obtained after dialysis and freeze-drying. The activator consists of EDC and NHS; Carboxymethyl starch: activator: PDSE = 0.1~1.5: 0.1~5.0: 0.1~10.0 weight ratio; EDC:NHS= 0.1~6:0.1~5.0 weight ratio; EDC is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and NHS is N-hydroxysuccinimide; PDSE is 2, 2′-(propane-2, 2-diylbis(sulfonamide))diethylamine; 2) Prepare an aqueous phase and an oil phase respectively, wherein the aqueous phase contains the ROS-responsive starch-based wall material PDSE-CMS, and the oil phase contains the pesticide and the cross-linking agent: The ROS-responsive starch-based wall material PDSE-CMS obtained in step 1) is dissolved in an aqueous solution containing a surfactant to serve as the aqueous phase; the concentration of the ROS-responsive starch-based wall material PDSE-CMS in the aqueous phase is 0.1-5.0 g / 100 ml, and the concentration of the surfactant in the aqueous solution containing the surfactant is 0.2-5.0 g / 100 ml; the surfactant is a mixture of fatty alcohol polyoxyethylene ether 23: sodium lauryl sulfate in a weight ratio of 1:1; The pesticide and 2, 4, 6-tris(4-formylphenoxy)-1, 3, 5-triazine as a cross-linking agent are dissolved in an organic solvent as an oil phase; the mass ratio of the pesticide to the cross-linking agent is 1-10:1; the amount ratio of the organic solvent to the pesticide in the oil phase is (10-80) mL: 1 g; the organic solvent is methyl salicylate; 3) The oil phase obtained in step 2) is added to the aqueous phase obtained in step 2) at a volume ratio of organic solvent to aqueous solution containing a surfactant of 1:50-100. After emulsification and cell disruption and dispersion, an O / W emulsion is obtained. The emulsion is then heated for cross-linking and curing to perform an interfacial polymerization reaction, thereby obtaining ROS-responsive functionalized starch-based drug-loaded nano / microspheres.

2. The method for preparing ROS-responsive functionalized starch-based drug-loaded nano / microspheres according to claim 1, characterized in that In the step 3): The emulsification step comprises: performing an emulsification treatment at a rotation speed of 10,000 to 20,000 rpm for 1 to 50 minutes; The cell disruption and dispersion is as follows: the dispersion time is 1 to 5 minutes under the condition of power 100W; The heating cross-linking curing is: reacting at a stirring speed of 200-500 rpm and 30-60° C. for 4-12 hours.

3. The method for preparing ROS-responsive functionalized starch-based drug-loaded nano / microspheres according to claim 2, characterized in that: The pesticide in step 2) is a fat-soluble pesticide; Fat-soluble pesticides include fat-soluble fungicides and fat-soluble insecticides.

4. The method for preparing ROS-responsive functionalized starch-based drug-loaded nano / microspheres according to claim 3, characterized in that: The fat-soluble fungicide is at least any one of the following: triadimefon, tebuconazole, propiconazole, difenoconazole, carbendazim, thiophanate-methyl, azoxystrobin, kresoxim-methyl, pyraclostrobin and the like; The fat-soluble insecticide is at least any one of the following: avermectin, beta-cypermethrin, fipronil, chlorpyrifos, indoxacarb and other insecticides.

5. The method for preparing ROS-responsive functionalized starch-based drug-loaded nano / microspheres according to claim 4, characterized in that: In the aqueous solution containing a surfactant, the concentrations of fatty alcohol polyoxyethylene ether 23 and sodium lauryl sulfate are both 0.2 g / 100 ml.

6. The ROS-responsive functionalized starch-based drug-loaded nano / microspheres obtained by any one of the methods of claims 1 to 5, characterized in that Any of the following scales: nanoscale, submicron scale, micron scale.

7. The use of the ROS-responsive functionalized starch-based drug-loaded nano / microspheres according to claim 6, characterized in that: It has the function of releasing pesticides in response to the presence of ROS; It can activate the plant's own antioxidant defense regulatory function.

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