Preparation method and application of lignin-based sustained-release microspheres with amino acid modification for promoting drug absorption and transmission

Abstract

The present application belongs to the field of pesticide preparation, and particularly relates to a preparation method of lignin-based sustained-release microspheres with amino acid modification for promoting drug absorption and conduction function and application thereof. The method comprises the following steps: first, esterification is performed to enhance the lipophilicity of the original carrier lignin; then, amino acid is grafted to enhance the hydrophilicity of the lignin, so as to reduce the surface tension of the lignin on the plant leaf surface, thereby enhancing the wetting and spreading properties of the lignin and the rainwater washing resistance of the lignin; and then, the pesticide, organic solvent and arginine modified esterified lignin are used as an organic phase, and an aqueous solution with a surfactant added is used as an aqueous phase; the oil phase and the aqueous phase are mixed and emulsified, and after the organic solvent is volatilized, the lignin microspheres loaded with the pesticide are obtained. The present application also simultaneously provides the use of the above microspheres for promoting drug absorption and conduction, and the microspheres are suitable for developing corresponding green agricultural carriers.

Description

Technical Field

[0001] This invention belongs to the field of pesticide formulations, specifically relating to a method for preparing amino acid-modified lignin-based sustained-release microspheres that promote drug absorption and conduction, and their application. Background Technology

[0002] To ensure high crop yields, pest and disease control primarily relies on rapid, efficient, economical, and convenient chemical methods. However, conventional pesticide formulations suffer from poor wetting and spreading properties on leaf surfaces due to surface tension, leading to pesticide aggregation, loss, or rolling off. This not only results in short-lasting effects and low utilization rates but also significant pesticide losses due to degradation, evaporation, surface runoff, and drift. Furthermore, the coarse particle size of existing formulations after dilution hinders absorption and transport within the crop to the target site, leading to insufficient efficacy. Repeated or excessive application poses risks to ecological safety and agricultural product quality. Therefore, the development of new pesticide technologies is urgently needed to address these issues. Nanocarriers, with their high dispersibility, high specific area, high loading capacity, and potential environmentally responsive decomposition, offer the potential to enable precise targeted pesticide application and minimize environmental risks by controlling off-target losses.

[0003] Lignin is a natural high-molecular-weight compound with a three-dimensional network structure, widely present in most plants, accounting for approximately 20%–30% of plant mass. Together with cellulose and hemicellulose, it forms the plant skeleton and accounts for about 30% of organic carbon in the biosphere. It is the second most abundant renewable resource in nature after cellulose, and its industrial production is mainly derived from pulp and paper making or biorefining. The structure of lignin itself (such as hydrophilic functional groups like carboxyl and phenolic hydroxyl groups, and hydrophobic functional groups like phenylpropane units) gives it excellent resistance to photodegradation and oxidation, as well as laccase-responsive properties. It is easily degraded in the natural environment, and using lignin as a wall material to prepare nano-controlled-release pesticides aligns with the principles of environmental friendliness and sustainable development.

[0004] Amino acids are the basic building blocks of living organisms, easily recognized and metabolized by plants, exhibiting extremely low residual toxicity, excellent biocompatibility, and environmental friendliness. They possess unique advantages as modifying groups in controlled-release drug delivery systems. *Nature Communications*, 16:6715 (2025.01), first reported that amino acid transporter-mediated nanoparticle carriers can autonomously enter living plants. The presence of plant cell walls and the lack of efficient pathways to deliver external substances into plant cells are core challenges facing the development of plant biotechnology and crop engineering. However, existing viral and bacterial transmission methods have limitations, and a few non-heavy metal nanoparticle delivery platforms require external force to penetrate tissues. This dependence limits the development of any high-throughput applications, especially considering the large number of individual plants that need to be handled in field or laboratory operations. Existing research has demonstrated a polyethylene glycol-block-poly(2-diisopropylaminoethyl methacrylate) (Asp / PDPA-NP) nanocarrier platform modified with aspartic acid (Asp). This platform utilizes amino acid transport proteins (AtAAP1 and AtLHT1) as receptors and, through protein-dependent endocytosis, can freely transport and release payloads to various tissue / cell types across different plant species within ≤10 minutes via simple spraying or co-culture. As a proof-of-concept, the efficacy of Asp / PDPA-NP loaded with the small molecule compound abscisic acid (ABA) in conferring drought resistance in plants was tested. This nanoparticle reduced the effective dose of ABA to 1 nanomolar (parts per million) and induced drought resistance activity in representative eudicotyledonous plants (soybeans) and monocotyledonous crops (maize). With its highly efficient delivery characteristics, Asp / PDPA nanoparticles hold promise as a potent carrier for various chemical substances and biomolecules in plant systems. The aforementioned literature verified that the modification of polymer nanocarriers with amino acid-aspartic acid can promote the cell penetration and absorption and conduction functions of nanoparticles in plants.

[0005] The journal *ACS Sustainable Chem. Eng*, 2024, 12, 5052−5060 (December 2024) reported a novel pH-responsive colloidal sphere formed by the remethylation reaction of amino acid-modified lignin. This research aims to solve the technical challenges of pH-triggered charge-reversal nanoparticles (pH-CRNPs), focusing on improving their precision and scalability for widespread application. Utilizing the unique properties of lignin, an abundant renewable aromatic macromolecule found in plants, lignin nanoparticles (LNPs) were successfully prepared using a one-pot method after chemical modification with three different amino acids (arginine (ARG), glutamic acid (GLU), and glycine (GLY)) followed by methylation. The amino acid modification significantly reduced the contact angle, enhancing the hydrophilicity of the carrier material. Arginine, in particular, showed the most significant improvement in hydrophilicity among the three amino acids: the aqueous phase contact angle decreased from 56.1° to 27.1°. Further methylation modification of the lignin amino acids resulted in tunable pH responsiveness and morphological changes. This method can be used to load drugs with different hydrophilicities and charges, thereby expanding the high-value applications of lignin in the biomedical field. However, this literature does not report on loading any drugs, including pesticides, nor does it cover the absorption and conduction properties after pesticide loading.

[0006] The journal *Green Chemistry*, 22, 6357–6371 (2020), reported on amino acid-functionalized polyampholytes as natural broad-spectrum antibacterial agents for highly effective personal protection. The study demonstrated that amino acid modification significantly enhances the antibacterial effect. A highly biocompatible amino acid-grafted enzymatically hydrolyzed lignin derivative (EHL-AAx) with both cationic and anionic groups was synthesized via nucleophilic substitution. In vitro bioassays showed that when the amino acid grafting amount ranged from 0 to 0.62 mmol g… -1 When the concentration was changed, the inhibition efficiency of EHL-AA-x against Gram-negative bacteria increased from 10% to 57% (at a concentration of 10 mg / mL). -1 It exhibits an inhibition efficiency of 99% against Gram-positive bacteria. This antibacterial efficiency is not only four times that of birch water-soluble lignin, but also comparable to lignin / nano-silver composite materials. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a method for preparing amino acid-modified lignin-based sustained-release microspheres that promote drug absorption and conduction, and their application.

[0008] To address the aforementioned problems, this invention provides a method for preparing amino acid-modified lignin-based sustained-release microspheres (i.e., arginine-modified esterified lignin drug-loaded microspheres), comprising the following steps:

[0009] 1) Dissolve lignin (alkali lignin) in an aprotic polar organic solvent, then add (dropwise) an acid-binding agent and an acylation reagent containing an organic alkene functional group (as an esterification material and to provide organic alkene groups), and stir the reaction at 30~50°C for 10~14h; after post-treatment, the resulting reactants are given esterified lignin (abbreviated as AM, brownish-yellow solid).

[0010] Lignin: acid-binding agent: acylation reagent containing organic olefin functional groups = 0.1~10 : 0.1~10 : 0.5~100 (preferably 1:1:0.5~1) by weight ratio;

[0011] 2) Arginine was selected as the amino acid;

[0012] Esterified lignin was dissolved in solvent I, arginine was added, and the mixture was reacted at 30-50°C for 10-14 h. The resulting reactants were then post-treated to obtain arginine-modified esterified lignin (abbreviated as AMA, a brown solid product, i.e., arginine-modified esterified lignin).

[0013] The esterified lignin:arginine has a weight ratio of 0.1~10:0.1~8; preferably 5:(3±0.5).

[0014] 3) Arginine-modified esterified lignin and pesticide (small molecule pesticide) are dissolved in solvent II as the oil phase; the weight ratio of arginine-modified esterified lignin to pesticide is 2~10:1, preferably 5:(1±0.2).

[0015] Use an aqueous solution of surfactant with a concentration of 0.1~1g / 100ml as the aqueous phase;

[0016] According to the mass ratio of arginine-modified esterified lignin to surfactant = 0.1~2:0.1~1, preferably 2:(1±0.2), the oil phase and the water phase are mixed and emulsified under ice bath conditions. Then, solvent II is evaporated to remove the emulsion, and arginine-modified esterified lignin drug-loaded microspheres (amino acid-modified lignin-based sustained-release microspheres) are obtained.

[0017] As an improvement to the preparation method of the amino acid-modified lignin-based sustained-release microspheres of the present invention, in step 1):

[0018] The acylation reagent containing an organic alkene functional group is any one of the following: methacrylic anhydride, methacryl chloride;

[0019] The acid-binding agent is at least one of the following: N-ethyldiisopropylamine, triethylamine, N,N-diisopropylmethylamine, N,N-diisopropyln-propylamine, 2,6-dimethylpyridine, or sulfonamide.

[0020] As a further improvement to the preparation method of the amino acid-modified lignin-based controlled-release microspheres of the present invention, the surfactant in step 3) is at least one of the following: polyvinyl alcohol, hexadecyltrimethylammonium bromide, sodium dodecylbenzenesulfonate, styrene, phenol-formaldehyde resin, polyoxyethylene sorbitan monooleate, polyoxyethylene polyoxypropylene block polyether, alkylphenol polyoxyethylene ether, octylphenol polyoxyethylene ether, nonylphenol polyoxyethylene ether, fatty alcohol polyoxyethylene, sulfonic acid esters, phthalamides, and organosilicones.

[0021] As a further improvement to the preparation method of the amino acid-modified lignin-based sustained-release microspheres of the present invention, in step 3):

[0022] Emulsification involves: first homogenization, then dispersion (cell disruption and dispersion).

[0023] Specifically: first homogenize at 16,000 ± 20,000 rpm for 1-3 minutes under ice bath conditions (using a high-shear emulsifier); then disperse under ice bath conditions for 1-5 minutes (power 100 ± 20 W).

[0024] The solvent II is removed by evaporation by magnetic stirring at 15℃~40℃ and 500~1500 rpm for 2 ± 0.5 h (preferably 25℃ and 1000 rpm).

[0025] As a further improvement to the preparation method of the amino acid-modified lignin-based controlled-release microspheres of the present invention, in step 3), the pesticide agent is a fat-soluble pesticide or a water-soluble pesticide.

[0026] Note: The pesticides of this invention are conventional fungicides and insecticides.

[0027] The fat-soluble fungicide is at least one of the following: iprodione, triadimefon, tebuconazole, propiconazole, difenoconazole, carbendazim, thiophanate-methyl, azoxystrobin, pyraclostrobin, pyraclostrobin, etc.

[0028] The water-soluble and fat-soluble insecticides mentioned are at least one of the following: abamectin, high-efficiency cypermethrin, fipronil, chlorpyrifos, indoxacarb, etc.

[0029] As a further improvement to the preparation method of the amino acid-modified lignin-based sustained-release microspheres of the present invention:

[0030] In step 1), lignin: aprotic polar organic solvent = 1 g / 20~30 ml, and the aprotic polar organic solvent is N,N-dimethylformamide;

[0031] In step 2), esterified lignin: solvent I = 1 g / 40~80 ml, and solvent I is tetrahydrofuran;

[0032] In step 3), arginine-modified esterified lignin: solvent II = 1 g / 20~50 ml; solvent II is dichloromethane.

[0033] As a further improvement to the preparation method of the amino acid-modified lignin-based sustained-release microspheres of the present invention:

[0034] The post-processing of step 1) is as follows: Centrifuge the obtained reaction mixture (8000~12000 rpm for 5~10 minutes to remove precipitate impurities), add deionized water to the supernatant to precipitate the product, and then centrifuge (8000~12000 rpm for 5~10 minutes); repeat the above steps of adding deionized water and centrifuging the supernatant obtained by centrifugation (repeated 2~4 times), and freeze-dry the solid obtained from the last centrifugation (vacuum freeze-drying) to obtain esterified lignin (abbreviated as AM, brownish-yellow solid);

[0035] The volume ratio of supernatant to deionized water is 1:1~2.

[0036] The post-treatment of step 2) is as follows: remove solvent I by rotary evaporation, dissolve the obtained concentrated product in dichloromethane, add saturated brine water to wash and remove the arginine that has not been completely reacted, concentrate the obtained organic phase (remove dichloromethane by rotary evaporation) to obtain arginine-modified esterified lignin.

[0037] This invention also provides amino acid-modified lignin-based sustained-release microspheres (arginine-modified esterified lignin drug-loaded microspheres) prepared using any of the above methods. The microspheres have a size of 1-200 nm.

[0038] The present invention also provides the use of amino acid-modified lignin-based sustained-release microspheres: promoting the absorption and conduction of drugs, that is, helping to expand the scope of drug action while improving efficacy, and therefore suitable for developing corresponding green agricultural carriers.

[0039] An improvement to the use of the amino acid-modified lignin-based sustained-release microspheres of the present invention: having the function of releasing drugs in response to laccase, it can be used to prepare a drug-targeted controlled release system for environments with high laccase concentrations. This system can quickly sense environmental changes and release drugs, thereby increasing the local drug concentration and achieving a rapid antibacterial effect.

[0040] Therefore, this invention relates to a method for preparing amino acid-modified lignin-based sustained-release microspheres with laccase-responsive and drug absorption-promoting functions. In this invention:

[0041] Step 1) Modify lignin by esterification reaction, so that organic alkene functional groups are grafted onto lignin molecules to obtain esterified lignin, thereby improving its lipophilicity.

[0042] Step 2) Dissolve the esterified lignin in an organic solvent and graft arginine via Michael addition to obtain arginine-modified esterified lignin (arginine-modified esterified lignin), thereby improving its hydrophilicity.

[0043] Step 3) Using pesticide, organic solvent, and arginine-modified esterified lignin as the organic phase and an aqueous solution with added surfactant as the aqueous phase, the oil phase and the aqueous phase are mixed and emulsified, and after the organic solvent is evaporated, lignin microspheres loaded with pesticide are obtained.

[0044] This invention designs a novel route for modifying lignin with amino acids. First, an esterification reaction is used to enhance the lipophilicity of the original carrier lignin. Then, amino acids are grafted to enhance its hydrophilicity and reduce its surface tension on plant leaves, thereby enhancing its wettability, spreadability, and resistance to rain washout. Subsequently, by reducing the particle size of the drug-loaded particles, the absorption and conduction performance in the plant is further enhanced. Combined with the release of laccase in the plant, the drug-loaded particles respond to release the drug, ultimately reducing pesticide dosage and increasing efficiency, and reducing environmental pressure.

[0045] In the process of inventing this invention, the following problems that needed to be solved were fully considered:

[0046] Although there are numerous patent documents and academic reports on the modification of lignin using arginine, existing research mainly focuses on the development of green and safe wood adhesives, biomedical antibacterial coatings, or hydrogels. In these existing technologies, the introduction of arginine primarily utilizes its positively charged guanidine group to achieve simple adhesion enhancement or surface contact bactericidal function. However, search results show that there are no reports on the application of arginine-modified lignin in pesticide controlled-release systems, particularly regarding the use of such materials to adjust the contact angle of pesticide solutions on crop leaves (wetting properties), improve the resistance of pesticide solutions to rain washout (adhesion), and enhance the absorption and transport properties of drugs within plants (systemic conductivity). Furthermore, in terms of synthetic strategies, existing reports mostly employ direct grafting or modification through other intermediates. To date, no preparation process has been reported that first constructs esterified lignin containing specific reaction sites (such as olefin double bonds) as a precursor through esterification modification (the purpose of esterification is to increase the lipophilicity of lignin), and then further modifies it with arginine grafting (to fine-tune and increase the hydrophilicity of lignin). This specific "stepwise dual modification" strategy has not been disclosed in existing technologies, which limits the flexibility and precision of functional regulation of existing lignin-based carrier materials, making it difficult to meet the needs of modern agriculture for efficient and precise delivery carriers.

[0047] To address the aforementioned issues, the inventors' research group conducted in-depth studies. Based on a comprehensive consideration of current research reports, they modified esterified lignin by grafting arginine onto the esterified lignin. This modification adjusts the carrier's amphiphilicity to adapt to leaf surfaces and utilizes its penetrating properties to facilitate the transport of small pesticide molecules (such as iprodione) through the plant cuticle, thus endowing the drug with systemic conductivity. A laccase-controlled release switch was introduced. In the event of *Botrytis cinerea* infection of crops, this degradation mechanism serves as a biotrigger for targeted pesticide release—when laccase acts on the lignin-based nanocarrier, the encapsulated pesticide is rapidly released at the infection site. This design aims to simultaneously achieve targeted delivery and slow release of pesticides, ultimately obtaining targeted, slow-release, and sustained control of diseases, while also reducing pesticide migration and loss in the environment, achieving the goal of "reduced pesticide use and increased efficacy."

[0048] In addressing the technical bottlenecks of existing pesticide formulations in practical agricultural production, such as low effective utilization, poor adhesion to crop leaves, susceptibility to rainwater runoff, and the high environmental risks caused by the non-degradability of traditional polymeric carrier materials leading to microplastic residues, this invention provides a simple and easy-to-prepare arginine-modified lignin pesticide carrier, along with its preparation method and applications. This carrier system not only possesses excellent leaf affinity and systemic conductivity but also enables intelligent response release to enzymes secreted by pathogenic fungi, thus constructing a new, efficient, safe, and environmentally friendly strategy for fungal disease management. The pesticide-loaded particle suspension provides protection simply by spraying during application. This invention systematically characterized the formation mechanism, morphology, sustained-release performance, and absorption and conductivity of the pesticide-loaded particles. Finally, this invention evaluated its protective ability against pathogenic fungi through pot experiments and comprehensively assessed the application safety of the system through zebrafish acute toxicity tests. This research is expected to provide a new strategy for efficient, safe, and sustainable fungal disease management in modern agricultural systems. This invention not only possesses the rapid-acting effect of external spraying for disease control but also exhibits a slow-release, sustained-release effect. Simultaneously, it reduces the migration and runoff characteristics of existing pesticides, thus better mitigating environmental risks and demonstrating significant technological advantages. Furthermore, the pesticide-carrying material of this invention has a slow-release function, high crop safety, and gradually decomposes in the environment, effectively avoiding environmental pollution problems caused by microplastic residues.

[0049] Compared with the prior art, the present invention has the following technical advantages:

[0050] 1. This invention uses lignin (alkali lignin), which is widely available, environmentally friendly, and biocompatible, as raw material. Taking advantage of its structural modifiability and biocompatibility, amino acids are grafted onto lignin-based controlled-release microspheres through a simple and mild chemical modification method.

[0051] 2. By modifying with arginine, the wetting, spreading and adhesion properties of the drug-loaded solution on the surface of crop leaves were significantly improved, reducing the loss due to droplet rolling, splashing and rain rinsing, thus improving drug utilization and reducing the impact on non-target organisms.

[0052] 3. The use of arginine-modified esterified lignin materials to prepare drug-loaded microspheres enhances the absorption and transport capacity of drugs, enabling effective control of diseases that invade below the leaf surface of plants, extending the protective range of drugs, and forming a protective system that combines internal and external protection.

[0053] 4. By physically encapsulating the drug and utilizing the inherent advantages of lignin-based materials in resisting UV degradation, the drug's degradation half-life is effectively protected and extended.

[0054] 5. The arginine-modified esterified lignin drug-loaded microspheres constructed in this invention have laccase release response characteristics, which can achieve rapid pesticide release against pathogens that release laccase invading plants, block the spread of pathogens, and improve the control effect.

[0055] 6. The lignin microspheres prepared according to the present invention have controllable particle size and drug loading, and can adapt to various spraying environments. They have considerable application prospects in the field of pesticide formulation, especially in the field of controlled-release formulation.

[0056] 7. This invention, combined with acute toxicity tests on zebrafish and pot experiments, demonstrates that the drug delivery system exhibits low toxicity to non-target organisms (such as zebrafish) and is safe for crop growth without causing phytotoxicity. Therefore, the system of this invention can significantly reduce the migration and runoff characteristics of existing pesticides, thereby reducing environmental risks to soil and water bodies.

[0057] 8. This invention uses natural polymer lignin as a base material. The modified material has good biodegradability in the environment and can be gradually broken down, effectively avoiding the microplastic residue and environmental pollution problems caused by traditional synthetic polymer carriers.

[0058] In summary, this invention addresses the technical bottlenecks of existing hydrophobic pesticide formulations, such as poor dispersibility in water, low bioavailability, and weak targeted delivery capabilities, as well as the difficulty in degradation and the generation of microplastic residues that pollute the environment associated with traditional synthetic polymeric pesticide carriers. The invention aims to provide a drug-loaded microsphere based on arginine-modified esterified lignin, its preparation method, and its applications. This invention constructs a pesticide delivery system with small particle size, high stability, and excellent plant affinity and systemic conductivity through specific chemical modifications of natural polymeric lignin, thereby achieving pesticide reduction with increased efficiency and green plant protection. This invention uses widely available, biodegradable lignin as the main material, preparing esterified lignin with olefin reaction sites through esterification modification, and obtaining stable drug-loaded microspheres after arginine modification. This invention aims to improve the targeted delivery capability of hydrophobic drugs, regulate the morphology and function of drugs on leaves, and enable effective control in environments where diseases can spread widely, thereby improving drug efficacy and achieving the goal of reducing pesticide use while increasing efficiency. This invention modifies existing esterified lignin with arginine, significantly reducing the particle size of the drug-loaded particles (compared to single-esterified lignin drug-loaded particles) by adjusting the amphiphilic balance of the polymer. Smaller particle size not only improves the physical stability of the dispersion but also increases the contact surface area of ​​the drug solution on crop leaves, thereby enhancing efficacy. This invention utilizes the characteristic of arginine as a basic unit of life (amino acid), endowing the carrier with excellent biocompatibility. The modified drug-loaded particles are easily recognized and absorbed by plant roots or leaves, significantly increasing the migration of pesticides within the plant (systemic conductivity). This characteristic allows the drug to overcome the limitations of the contact site, extending the protective range and effectively controlling diseases that are highly concealed and easily spread over large areas. Furthermore, this invention uses lignin, a natural and renewable resource, as the main component, combined with bio-derived arginine for modification. After completing its drug delivery task, the entire carrier material can be gradually broken down and metabolized by microorganisms in the natural environment, ultimately degrading into harmless products. This fundamentally solves the microplastic residue problem caused by traditional non-degradable polymer carriers, meeting the needs of green agricultural development. This drug delivery system improves pesticide utilization in multiple dimensions by enhancing the dispersibility of hydrophobic drugs, regulating their spread on leaves, strengthening systemic transport, and providing long-lasting sustained release. While ensuring control efficacy, it significantly reduces pesticide dosage, lowering agricultural production costs and environmental toxicity risks. This preparation strategy combines the advantages of ease of operation and superior performance, overcoming the limitations of existing lignin modification technologies that suffer from single-function applications and poor drug delivery performance, providing a new technological pathway for developing novel compound-efficacy pesticide formulations. This research is expected to provide a new strategy for efficient, safe, and sustainable disease management in modern agricultural systems. Attached Figure Description

[0059] Figure 1 This is a Fourier transform infrared spectrum;

[0060] Figure 1 middle:

[0061] (a) Fourier transform infrared spectra of alkali lignin, esterified lignin obtained in step (1) of Example 1, and arginine-modified esterified lignin obtained in step (2) of Example 1.

[0062] (b) Fourier transform infrared spectra of isopropionate, isopropionate esterified lignin microspheres obtained in Comparative Example 1, and isopropionate arginine-modified esterified lignin microspheres obtained in step (3) of Example 1.

[0063] Figure 2 Image from a scanning electron microscope (SEM);

[0064] Figure 2 middle:

[0065] a: Scanning electron microscope image of the isourea-arginine-modified lignin microspheres obtained in step (3) of Example 1;

[0066] b: Scanning electron microscope image of the isourea-esterified lignin microspheres obtained in Comparative Example 1.

[0067] Figure 3 The graph shows the release rate curves of the isopropyl urea-arginine-modified lignin microspheres in Example 1 at different concentrations of laccase.

[0068] Figure 4 The following are examples: the isopropionyl arginine-modified lignin microspheres obtained in Example 1, the isopropionyl arginine-modified lignin microspheres obtained in Comparative Example 1, the commercially available isopropionyl suspending agent, and the contact angle of water on different plant leaves.

[0069] Figure 5 The rain erosion resistance of the isopropionyl arginine-modified lignin microspheres (Ipr@AMA) obtained in Example 1, the isopropionyl arginine-modified lignin microspheres (Ipr@AM) obtained in Comparative Example 1, and the commercially available isopropionyl suspending agent (Ipr SC) on plant leaves.

[0070] Figure 6 This study presents the in vivo inoculation efficacy experiment of the isopropylurea arginine-modified lignin microspheres and the commercial isopropylurea suspension obtained in Example 1 on pepper plants.

[0071] Figure 6 middle:

[0072] (a): CK represents the leaf disease status of the control group sprayed with deionized water; Ipr SC represents the leaf disease status of the treatment group sprayed with commercial iprodione suspension; Ipr@AMA represents the leaf disease status of the treatment group sprayed with iprodione arginine-modified lignin-loaded microspheres.

[0073] (b): Leaf disease index of the treatment group sprayed with commercial iprodione suspension on day 0, day 7 and day 14 after spraying, and leaf disease index of the treatment group sprayed with iprodione arginine-modified lignin-loaded microspheres.

[0074] Figure 7 This is a visualization (laser confocal image) of the delivery and distribution of arginine-modified esterified lignin microspheres loaded with Nile Red tracer in pepper seedlings in Example 2.

[0075] Figure 8 The images show the drug delivery and dose distribution of the iprodione arginine-modified lignin microspheres obtained in Example 1, the iprodione esterified lignin microspheres obtained in Comparative Example 1, and the commercial iprodione suspension in pepper seedlings.

[0076] Figure 8 middle:

[0077] (a) Drug delivery volume at different sites;

[0078] (b) Drug residue rate at different sites.

[0079] Figure 9 The acute toxicity of different concentrations of the isopropyl arginine-modified lignin microspheres, commercially available isopropyl arginine suspension concentrate, and isopropyl arginine technical grade obtained in Example 1 to adult zebrafish is shown. Detailed Implementation

[0080] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0081] The proton nuclear magnetic resonance spectrometer used in this invention is a Bruker AVANCE III 400; the transmission electron microscope is an HT-7700 field emission transmission electron microscope (Hitachi, Japan); the thermal field emission scanning electron microscope is a Zeiss G300; the liquid chromatograph is a Shimadzu LC-10AT; the particle size analyzer is a Nano-ZS90 laser particle size analyzer; and the contact angle meter is a Shanghai Zhongchen XG-CAM.

[0082] Alkali lignin is available from Sigma-Aldrich (471003-100).

[0083] Iprodione commercial suspension concentrate is available from Qingdao Hainan Biotechnology Co., Ltd., pesticide registration number PD20141643;

[0084] Iprodione technical grade can be purchased from Tianjin Xisien Biochemical Technology Co., Ltd., model number D-24855.

[0085] All water used is deionized.

[0086] In this invention, the stirring speed, which is not explicitly specified, is approximately 200~400 rpm.

[0087] Example 1: A method for preparing arginine-modified esterified lignin sustained-release drug-loaded microspheres (arginine-modified esterified lignin drug-loaded microspheres, amino acid-modified lignin-based sustained-release microspheres), comprising the following steps:

[0088] (1) Preparation method of esterified lignin:

[0089] Add 2 g of alkali lignin (AL) to 50 ml of N,N-dimethylformamide and heat at 90 °C for 1 h under a nitrogen atmosphere until the alkali lignin is completely dissolved. Then lower the temperature to 30 °C. Weigh 2 g of triethylamine as an acid-binding agent and add it dropwise (approximately 1 minute each time), followed by stirring for 30 minutes. Add 2 g of methacrylamide chloride dropwise (approximately 2 minutes each time) to the system, and then react overnight (approximately 12 h) at 50 °C with stirring.

[0090] After the reaction was completed, the mixture was centrifuged at 8000 rpm for 10 min. The supernatant obtained by centrifugation was then mixed with water (the volume of water was twice that of the supernatant) to precipitate the product. The precipitation time was 5 min. Then, the mixture was centrifuged at 8000 rpm for 10 min. The supernatant obtained by centrifugation was repeated with water addition for precipitation and centrifugation (repeated 2-4 times). The solid obtained from the last centrifugation was freeze-dried under vacuum in a freeze dryer (frozen at -50℃ to constant weight) to obtain about 1.1 g of brownish-yellow solid (esterified lignin, abbreviated as AM), with a yield of 55%.

[0091] (2) Preparation method of arginine-modified esterified lignin:

[0092] Dissolve 500 mg of esterified lignin in 30 ml of tetrahydrofuran, then add 300 mg of arginine and react overnight (about 12 h) under gentle stirring at 40 °C.

[0093] After the reaction was completed, tetrahydrofuran was evaporated by rotary evaporation. The concentrated product was dissolved in dichloromethane (about 20 ml), and saturated brine was added for three separate extractions (about 20 ml of saturated brine was used each time). The organic phase was then rotary evaporated (to remove dichloromethane) to obtain arginine-modified esterified lignin (abbreviated as AMA, a brown solid product).

[0094] The specific reaction equation is shown below:

[0095]

[0096] (3) Preparation of arginine-modified esterified lignin-loaded microspheres loaded with the fungicide iprodione:

[0097] Take 50 mg of arginine-modified esterified lignin and dissolve it with 10 mg of iprodione in 1 ml of dichloromethane as the oil phase.

[0098] Take 5 ml of 0.5% (i.e., 0.5 g / 100 ml) polyvinyl alcohol solution as the aqueous phase.

[0099] The oil and aqueous phases were mixed and homogenized for two minutes at 16,000 rpm using a high-speed homogenizer under ice bath conditions. Then, the mixture was run for two minutes using a cell disruptor with a 2-second start-4-second pause frequency. Afterward, the mixture was magnetically stirred at 1000 rpm at 25°C for 2 hours to volatilize dichloromethane, thereby preparing arginine-modified esterified lignin drug-loaded microspheres (Ipr@AMA, i.e., isopropyl urea arginine-modified esterified lignin microspheres). The drug-loaded particles exhibited good stability, and the drug could be effectively loaded without any drug crystal precipitation or visible sedimentation.

[0100] The average particle size of the drug-loaded microspheres prepared in Example 1 was approximately 180 nm, as determined by Malvern Zetasizer Nano ZS90. Figure 2 As shown in a.

[0101] Example 2: A method for preparing arginine-modified esterified lignin-loaded fluorescent tracer microspheres, comprising the following steps:

[0102] (1) Preparation method of esterified lignin:

[0103] Same as step (1) in Example 1.

[0104] (2) Preparation method of arginine-modified esterified lignin:

[0105] Same as step (2) in Example 1.

[0106] (3) Preparation of arginine-modified esterified lignin-loaded microspheres loaded with the fluorescent tracer Nile Red:

[0107] Replace “10 mg iprodione” with “10 mg Nile red” in step (3) of Example 1, and the rest is the same as step (3) of Example 1; to obtain fluorescent tracer microspheres (Nile@AMA).

[0108] The average particle size of the fluorescent tracer microspheres was measured to be approximately 196 nm using the Malvern Zetasizer Nano ZS90.

[0109] Note: In Example 2, no pesticide was loaded, but the fluorescent tracer Nile Red was loaded. The purpose was to detect whether the prepared arginine-modified esterified lignin had the ability to be absorbed and translocated in plants.

[0110] Example 3: A method for preparing arginine-modified esterified lignin sustained-release drug-loaded microspheres, comprising the following steps:

[0111] (1) Preparation method of esterified lignin:

[0112] The triethylamine used as the acid-binding agent was replaced with N-ethyldiisopropylamine, and the amount remained unchanged at 2 g; the rest was the same as step (1) of Example 1.

[0113] (2) Preparation method of arginine-modified esterified lignin:

[0114] Same as step (2) in Example 1.

[0115] (3) Preparation of arginine-modified esterified lignin-loaded microspheres loaded with the fungicide iprodione

[0116] Same as step (3) in Example 1.

[0117] The average particle size of the drug-loaded microspheres was determined to be approximately 180 nm using Malvern Zetasizer Nano ZS90.

[0118] Example 4: A method for preparing arginine-modified esterified lignin sustained-release drug-loaded microspheres, comprising the following steps:

[0119] (1) Preparation method of esterified lignin:

[0120] Replace “2 g methacryloyl chloride” with “1 g methacrylic anhydride”; the rest is the same as step (1) of Example 1.

[0121] (2) Preparation method of arginine-modified esterified lignin:

[0122] Same as step (2) in Example 1.

[0123] (3) Preparation of arginine-modified esterified lignin-loaded microspheres loaded with the fungicide iprodione:

[0124] Same as step (3) in Example 1 above.

[0125] The average particle size of the drug-loaded microspheres was determined to be approximately 186 nm using Malvern Zetasizer Nano ZS90.

[0126] Example 5

[0127] The concentration of the polyvinyl alcohol solution in step (3) of Example 1 was adjusted (as shown in Table 1 below), while the rest remained the same as in Example 1, so as to compare the particle size and distribution of the prepared arginine-modified esterified lignin drug-loaded microspheres (as shown in Table 1 below).

[0128] Table 1

[0129]

[0130] According to Table 1:

[0131] When the concentration of polyvinyl alcohol is 0.5%, arginine-modified esterified lignin drug-loaded microspheres with optimal particle size and distribution can be obtained, with a particle size of less than 200 nm.

[0132] Comparative Example 1: Preparation of drug-loaded microspheres (Ipr@AM) directly used as carriers after modification with esterified lignin

[0133] The difference from Example 1 is that step (2) is removed, that is, the esterified lignin obtained in step (1) is used to replace the arginine-modified esterified lignin directly in step (3) to obtain isopropionate esterified lignin microspheres (Ipr@AM).

[0134] According to measurements using Malvern's Zetasizer Nano ZS90, the average particle size of these drug-loaded microspheres is approximately 233 nm. Figure 2 As shown in b,

[0135] Comparative Example 2

[0136] The amount of arginine used in step (2) of Example 1 was changed to 600 mg, that is, the amount of arginine added was increased, and the remaining steps were the same as steps (1) to (2) of Example 1, to obtain arginine-modified esterified lignin.

[0137] The arginine-modified esterified lignin was prepared according to the method in step (3) of Example 2. It was found that after the dichloromethane evaporated, the stability of the drug-loaded particles was poor, the drug could not be effectively loaded, and drug crystals precipitated out, with visible precipitation.

[0138] Comparative Example 3

[0139] The amount of pesticide iprodione added in step (3) of Example 1 was increased from 10 mg to 30 mg, that is, the amount of pesticide small molecules added was increased. The remaining steps were the same as in Example 1. After the dichloromethane evaporated, it was found that the drug could not be effectively loaded and drug crystals precipitated out, which were visible to the naked eye.

[0140] Comparative Example 4, compared to Example 1, makes the following changes:

[0141] Cancel step (1);

[0142] Change step (2) to:

[0143] Dissolve 500 mg of lignin in 30 ml of tetrahydrofuran, then add 300 mg of arginine and react overnight under gentle stirring at 40°C.

[0144] The post-treatment is the same as step (2); the result is that after rotary evaporation of the organic phase, the result is still lignin (AL, brown solid powder).

[0145] Note: Since there are no reaction sites on lignin, arginine cannot participate in the reaction, so what is obtained is still the starting material.

[0146] Comparative Example 5, compared to Example 1, the following changes were made:

[0147] The post-processing in step (1) was modified to follow the method described in the reference (ACS Nano 2024, 18, 14, 10031–10044), namely: the mixture after the reaction was precipitated in water, centrifuged at 8000 rpm for five minutes, and the solid was separated. The solid obtained by centrifugation was repeatedly dissolved in chloroform and precipitated three times in deionized water to remove impurities. The product was freeze-dried under vacuum in a freeze dryer to obtain a brownish-yellow solid (Ipr@AM) with a yield of 55%, consistent with the yield of Example 1. However, the use of chloroform as a solvent is harmful to health; the process improvement of this invention is more environmentally friendly and safer.

[0148] Experiment 1: Structural Characterization

[0149] To verify the successful modification of esterified lignin and arginine-modified esterified lignin, infrared spectroscopy was performed on the alkali lignin raw material, the esterified lignin obtained in step (1) of Example 1, and the arginine-modified esterified lignin obtained in step (2) of Example 1. Figure 1 As shown in (a): the benzene ring bending vibration in the raw material alkali lignin is at 1510 cm. -1 It was observed at 1738 cm; while esterified lignin was observed at 1738 cm. -1 A strong signal was observed at the C=O position of the ester, and at approximately 3500 cm⁻¹ -1 The characteristic hydroxyl group at 947 cm is weakened. -1 The peak at [value missing] is attributed to the bending vibration of the acryloyl group. These characteristic peaks indicate successful grafting of the methacrylate group. Subsequently, the olefin of the esterified lignin and the amino group on arginine underwent a Michael addition reaction, followed by further amination based on esterification. Arginine-modified esterified lignin reached 947 cm⁻¹. -1 The decrease in peak intensity at 3300–3500 cm⁻¹ is attributed to the consumption of acryloyl C=C. -1 The double peak at 1600–1680 cm⁻¹ is attributed to the stretching vibration of the NH bond. -1The peak is attributed to the C=N stretching vibration of the guanidine group, indicating that the reaction successfully synthesized arginine-modified esterified lignin.

[0150] To verify the successful preparation of lignin-based drug-loaded sustained-release microspheres supported on iprodione, infrared spectroscopy was used to characterize the drug-loaded microspheres obtained in Example 1 and Comparative Example 1. Figure 1 As shown in (b): at 1738 cm -1 Stretching vibrations of the carbonyl group can be observed at 800 cm⁻¹. -1 The observation of C-Cl bond stretching vibrations proves that Ipr was successfully loaded into esterified lignin and arginine-modified esterified lignin nanocarriers.

[0151] Figure 2 Image a is a scanning electron microscope image of the iprodione arginine-modified esterified lignin microspheres (iprodione-loaded arginine-modified esterified lignin-based drug-loaded sustained-release microspheres) prepared in Example 1. It can be seen that the drug-loaded microspheres are regular spherical in shape, with a relatively smooth surface and an average size of about 180 nm. Figure 2 b represents the isourea-coated lignin microspheres (loaded isourea-coated lignin microspheres) obtained in Comparative Example 1, which have a relatively rough surface and an average size of approximately 233 nm.

[0152] Experiment 2: Laccase-responsive sustained-release effect of drug-loaded microspheres obtained in Example 1

[0153] The drug-loaded microspheres obtained in Example 1 were first freeze-dried at -40°C for 12 hours to obtain dried drug-loaded microspheres. Then, the following procedure was performed: the release behavior of iprodione from the drug-loaded microspheres was studied using dynamic dialysis. 0.1 g of the dried drug-loaded microspheres were dispersed in 2 ml of 0.7% (v / v) T-80 solution containing laccase (0, 10, 20 U / g), and transferred to a dialysis bag with a molecular weight cutoff of 1000. The dialysis bag was immersed in a brown bottle containing 100 ml of 0.7% T-80 solution and shaken on a constant temperature shaker at 25°C and 150 rpm. A 1 ml sample was periodically taken from the dialysis bag, and 1 ml of 0.7% T-80 solution was promptly added to the dialysis bag (i.e., an equal volume of 0.7% TW-80 solution was added after each sample). After filtering the sample through a 0.45 µm aqueous filter membrane, the iprodione content in the sample was determined by high-performance liquid chromatography (HPLC). Plot the iprodione release curve by plotting time on the x-axis and the cumulative percentage of iprodione release on the y-axis, repeating the process three times and taking the average value.

[0154] HPLC determination of iprodione content: The analytical column was an Agilent ZORBAX SB-C18 column (4.6×250mm, 5.0μm), the mobile phase was V(methanol):V(water) = 70:30, the flow rate was 1.0 mL / min, the detector was a UV detector with a detection wavelength of 280 nm, the injection volume was 20 μL, the column temperature was 30 ℃, and the standard curve method was used for quantification.

[0155] That is, the weakly basic response release performance of the drug-loaded microspheres was measured, and the results are as follows: Figure 3 As shown. From Figure 3 It can be seen that the release rates of iprodione-loaded microspheres prepared at different laccase concentrations showed significant differences. The release rate order was: 0 U / g < 10 U / g < 20 U / g. The different drug release rates exhibited by the microspheres prepared under different laccase concentrations enabled controlled and sustained release of the pesticide from the loaded particles.

[0156] The results showed that the drug release rate was related to the concentration of laccase, and the drug-loaded microspheres exhibited different drug release rates.

[0157] As is common knowledge, the active ingredient in pesticide iprodione does not have a sustained-release effect.

[0158] Experiment 3: Contact Angles of Different Plant Leaf Surfaces

[0159] The iprodione-loaded lignin microspheres (iprodione arginine-modified lignin microspheres) prepared in Example 1 were designated Ipr@AMA, the iprodione-loaded lignin microspheres prepared in Comparative Example 1 were designated Ipr@AM, and the commercially available iprodione suspension was designated Ipr SC. The wettability and spreading properties of the suspensions on leaf surfaces were studied using a contact angle meter (deionized water served as a blank control). Cucumber, tomato, pepper, and wheat leaves were selected, cut, and fixed onto glass slides. Iprodione suspensions (Ipr SC), Ipr@AM, and Ipr@AMA were diluted with deionized water to an iprodione concentration of 100 mg / L. 10 μL of the solution was dropped onto different leaf surfaces, and the contact angle was recorded after 60 seconds.

[0160] The results are as follows Figure 4The results show that the contact angle of Ipr@AMA on both hydrophilic (cucumber and tomato) and hydrophobic (wheat and pepper) leaves is smaller than that of water, Ipr SC, and Ipr@AM obtained in Comparative Example 1. The reduction in contact angle is particularly pronounced on hydrophilic leaves of cucumber and tomato, resulting in better wetting and spreading properties. Therefore, it can be concluded that arginine modification and the use of surfactant PVA (polyvinyl alcohol) to adjust particle size both contribute to reducing the contact angle of Ipr@AMA. This reduction in contact angle increases the contact area between the droplet and the leaf surface, thereby improving the wettability and retention of the leaf, and providing support for further absorption and transport of the drug within the plant.

[0161] Experiment 4: Resistance to rain erosion

[0162] The iprodione arginine-modified lignin microspheres prepared in Example 1 were designated Ipr@AMA, the iprodione esterified lignin microspheres prepared in Comparative Example 1 were designated Ipr@AM, and the commercially available iprodione suspension was designated Ipr SC. Their anti-rain washout effects on pepper leaves were compared. 5 mL of the solution sample (iprodione effective concentration of 1000 mg / L, diluted with deionized water) was sprayed onto pepper leaves. The leaves were then allowed to air dry at room temperature. Subsequently, the leaves were divided into two parts along the veins. One part of the leaf was fixed on a 60° angled wooden block, and 5 mL of deionized water was used to simulate rain for 60 seconds. The leaves were then allowed to air dry at room temperature. Samples were taken from the leaves before and after the rain washout using a punch and placed in 1 mL centrifuge tubes. 1 mL of acetonitrile was added for extraction, and the residue on the leaf surface was analyzed using high-performance liquid chromatography (HPLC). The results are as follows: Figure 5 As stated above.

[0163] The HPLC method for determining the content of iprodione is the same as described in Experiment 1.

[0164] The results are as follows Figure 5 As shown, the results indicate that the arginine-modified esterified lignin-loaded microspheres exhibit higher resistance to rainwater runoff than both esterified lignin-loaded microspheres and commercially available iprodione suspensions, demonstrating that the arginine-modified esterified lignin-loaded microspheres of this invention possess excellent resistance to rainwater runoff.

[0165] Experiment 5: Comparison of protective effects in live inoculation experiments

[0166] The arginine-modified lignin microspheres of iprodione prepared in Example 1 were designated Ipr@AMA, and the commercially available iprodione suspension concentrate was designated Ipr SC. The in vivo fungicidal activity of Ipr@AMA and its positive control, Ipr SC, against *Botrytis cinerea* was tested using pepper leaves as experimental material. Healthy pepper plants approximately two months old were selected and sprayed with Ipr@AMA and Ipr SC at a concentration of 300 μg / mL (both diluted with deionized water). These plants were then cultured at 25°C, 70% humidity, and a 16h / 8h diurnal cycle, and inoculated with *Botrytis cinerea* at three time points: day 0, day 7, and day 14. After 48 hours of culture at 25°C, the diameter of lesions was observed, and the results were statistically analyzed using the cross-multiplication method. Deionized water spraying served as a blank control. Each treatment was repeated three times.

[0167] .

[0168] The results are as follows Figure 6 As shown, the results indicate that the arginine-modified esterified lignin-loaded microspheres loaded with iprodione exhibit better control effects than commercially available iprodione suspensions, confirming that the arginine-modified esterified lignin-loaded microspheres of the present invention have sustained antifungal activity.

[0169] Experiment 6: Fluorescence Tracing Experiment

[0170] The arginine-modified esterified lignin fluorescent tracer microspheres loaded with Nile red fluorescent tracer prepared in Example 2 were designated Nile@AMA. By applying Nile@AMA to the surface of plant leaves, the distribution of Nile red fluorescence in different parts of the plant was observed using a fluorescence transmission microscope.

[0171] Leaf treatment: 500 mg / L Nile@AMA was applied to pepper leaves. After treatment, the plants were cultured in a constant temperature and light incubator (25℃, 16h / 8h light / dark cycle, 70% humidity) for 24h. The distribution of Nile@AMA in the leaves, stems and root cross sections was observed by fluorescence confocal microscopy.

[0172] Meanwhile, untreated chili peppers were used as a blank control.

[0173] like Figure 7 As shown, red fluorescence can be seen in the roots, stems, and leaves of the plant, proving that arginine-modified esterified lignin has a significant absorption and transport capacity as a carrier.

[0174] Experiment 7. Drug Absorption, Transport, and Content Distribution

[0175] The isopropionyl arginine-modified lignin microspheres prepared in Example 1 are designated as Ipr@AMA, the isopropionyl arginine-modified lignin microspheres prepared in Comparative Example 1 are designated as Ipr@AM, and the commercially available isopropionyl suspending agent is designated as Ipr SC. Their residual distribution in peppers is compared.

[0176] Ipr@AM, Ipr@AMA, and Ipr SC were diluted to 500 ppm. 0.2 ml of each medium leaf was evenly applied to each pepper seedling, and 0.5 mg of iprodione was applied to each seedling. After culturing the treated plants in a constant temperature and light incubator (25℃, 16h / 8h light / dark cycle, 70% humidity) for 24 h, samples were taken from the upper leaves, lower leaves, medium leaves (treated leaves), stems, and roots of the pepper seedlings for residue analysis.

[0177] The sample pretreatment steps are as follows: Take 0.5 g of sample, grind it with liquid nitrogen, and transfer it to a 50 ml centrifuge tube containing 4 ml of acetonitrile. Extract by sonication for 10 minutes. Then add 1 g of sodium chloride to the centrifuge tube and vortex for 1 minute. Centrifuge the mixture at 8000 rpm for 10 minutes. Take 1 ml of the supernatant and transfer it to a 2 ml centrifuge tube containing 50 mg of PSA and 100 mg of anhydrous magnesium sulfate. Vortex for 30 seconds and centrifuge at 12000 rpm for 5 minutes. After filtering the supernatant through an organic filter membrane, measure the residue using high-performance liquid chromatography (HPLC).

[0178] The results are as follows Figure 8 As shown, both Ipr@AM and Ipr@AMA nanocarriers exhibited high residual amounts and transfer rates in both lower and upper leaves. Among them, Ipr@AMA showed higher distribution amounts and transfer rates than Ipr@AM, indicating that arginine-modified carriers can effectively improve the absorption and translocation performance of pesticides in plants.

[0179] As is common knowledge, the pesticide iprodione itself has no systemic properties.

[0180] Experiment 8: Comparison of acute toxicity in zebrafish

[0181] The arginine-modified lignin microspheres prepared in Example 1 are designated as Ipr@AMA, the commercially available Ipr suspension is designated as Ipr SC, and the technical grade Ipr is designated as Ipr TC. To study the safety of lignin-based nanocarriers to organisms, the acute toxicity of different samples to zebrafish was measured, and the safety differences were compared.

[0182] Adult zebrafish were raised for more than 7 days in a laboratory environment at 27°C and a photoperiod of 14 / 10 h, with a mortality rate remaining below 5%. The zebrafish underwent intestinal cleansing (feeding stopped) 24 hours before the start of the experiment. During the experiment, 10 zebrafish were randomly selected from each treatment and exposed to different concentrations of Ipr SC, Ipr TC, and Ipr@AMA. Clean water was used as a control. The number of zebrafish deaths was counted at 24 h, 48 h, 72 h, and 96 h. Each treatment was replicated three times.

[0183] Figure 9 This is the result of the biosafety assessment for zebrafish. The results show that compared to iprodione technical and commercially available iprodione suspension concentrate, the iprodione arginine-modified esterified lignin microspheres of this invention reduce acute toxicity in zebrafish and are more biofriendly. The results indicate that the iprodione arginine-modified esterified lignin microspheres of this invention have good safety during use.

[0184] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. An amino acid-modified lignin-based controlled-release microsphere, characterized in that: The preparation method of amino acid-modified lignin-based sustained-release microspheres includes the following steps: 1) Dissolve lignin in an aprotic polar organic solvent, then add an acid-binding agent and an acylation reagent containing an organic alkene functional group, and stir the reaction at 30~50°C for 10~14 h; the resulting reactants are then post-treated to obtain esterified lignin. Lignin: acid-binding agent: acylation reagent containing organic olefin functional groups = 0.1~10 : 0.1~10 : 0.5~100 by weight ratio; The acylation reagent containing an organic alkene functional group is any one of the following: methacrylic anhydride, methacryl chloride; The acid-binding agent is at least one of the following: N-ethyldiisopropylamine, triethylamine, N,N-diisopropylmethylamine, N,N-diisopropyln-propylamine, and 2,6-dimethylpyridine. 2) Arginine was selected as the amino acid; Esterified lignin was dissolved in solvent I, arginine was added, and the mixture was reacted at 30-50°C for 10-14 h. The resulting reactants were then post-treated to obtain arginine-modified esterified lignin. The weight ratio of the esterified lignin to arginine is 0.1~10 : 0.1~8. 3) Arginine-modified esterified lignin and pesticide are dissolved in solvent II as the oil phase; the weight ratio of arginine-modified esterified lignin to pesticide is 2~10:1; the pesticide is any one of the following: iprodione, triadimefon, tebuconazole, propiconazole, difenoconazole; Use an aqueous solution of surfactant with a concentration of 0.1~1g / 100ml as the aqueous phase; The oil and aqueous phases were mixed and emulsified under ice bath conditions, and then solvent II was evaporated to obtain arginine-modified lignin-loaded microspheres.

2. The amino acid-modified lignin-based controlled-release microspheres according to claim 1, characterized in that... The surfactant in step 3) is at least one of the following: polyvinyl alcohol, hexadecyltrimethylammonium bromide, sodium dodecylbenzenesulfonate, polyoxyethylene sorbitan monooleate, polyoxyethylene polyoxypropylene block polyether, alkylphenol polyoxyethylene ether, octylphenol polyoxyethylene ether, nonylphenol polyoxyethylene ether, and fatty alcohol polyoxyethylene.

3. The amino acid-modified lignin-based controlled-release microspheres according to claim 2, characterized in that... In step 3): Emulsification involves first homogenizing, then dispersing. Solvent II was removed by evaporation by magnetic stirring at 15℃~40℃ and 500~1500 rpm for 2 ± 0.5 h.

4. The amino acid-modified lignin-based controlled-release microspheres according to claim 3, characterized in that: In step 1), lignin: aprotic polar organic solvent = 1 g / 20~30 ml, and the aprotic polar organic solvent is N,N-dimethylformamide; In step 2), esterified lignin: solvent I = 1 g / 40~80 ml, and solvent I is tetrahydrofuran; In step 3), arginine-modified esterified lignin: solvent II = 1 g / 20~50 ml; solvent II is dichloromethane.

5. The amino acid-modified lignin-based controlled-release microspheres according to any one of claims 1 to 4, characterized in that: The post-processing of step 1) is as follows: centrifuge the obtained reactants, add deionized water to the supernatant to precipitate the product, and then centrifuge; repeat the above steps of adding deionized water and centrifuging on the supernatant obtained by centrifugation, and freeze-dry the solid obtained by the last centrifugation to obtain esterified lignin. The volume ratio of supernatant to deionized water is 1:1~2. The post-treatment of step 2) is as follows: remove solvent I by rotary evaporation, dissolve the obtained concentrated product in dichloromethane, add saturated brine water to wash away the arginine that has not been completely reacted, and concentrate the obtained organic phase to obtain arginine-modified esterified lignin.

6. The use of the amino acid-modified lignin-based controlled-release microspheres as described in any one of claims 1 to 5, characterized in that: Used to promote drug absorption and delivery.

7. The use of the amino acid-modified lignin-based controlled-release microspheres according to claim 6, characterized in that: It has the function of releasing drugs in response to laccase.

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