A pH‐responsive vanillin‐Schiff base microcapsule for controlled release of pyraclostrobin

Abstract

BACKGROUND

Environmentally responsive microcapsules hold great promise for sustainable pest management by improving efficacy, lowering dosage and enhancing environmental compatibility. Herein, a pH‐responsive pyraclostrobin (Pyr)‐loaded microcapsule (Pyr@PU) with an isocyanate core and vanillin‐Schiff base polyurethane shell were successfully fabricated via interfacial polymerization.

RESULTS

The obtained microcapsules exhibited high loading capacity (80.89%) and encapsulation efficiency (96.17%), indicating excellent drug loading and encapsulation performance. In vitro release experiments confirmed that the Pyr@PU microcapsules exhibited prominent pH‐responsive release behavior, with a higher cumulative Pyr release (82.81%) at pH 4.0 after 144 h. Meanwhile, the prepared Pyr@PU microcapsules possessed excellent photostability, favorable leaf wettability and superior rainfastness. Bioactivity assays revealed that the microcapsules exerted favorable antifungal effects. At 200 μg mL⁻¹, Pyr@PU provided protective and curative efficacy of 79.62% and 67.59% against kiwifruit pathogens, as well as 57.90% and 37.89% on rice leaves, respectively. Biosafety evaluation showed that Pyr@PU reduced the 96‐h acute toxicity to zebrafish by 2.3‐fold and displayed no obvious adverse effects on rice seed germination.

CONCLUSION

The fabricated pH‐responsive Pyr@PU microcapsule exhibits superior UV resistance, leaf adhesion, rainfastness, antifungal efficacy, and biocompatibility. As a promising intelligent controlled‐release platform for pesticides, Pyr@PU provides both control efficacy and environmental compatibility, showing great potential for application in sustainable agriculture. © 2026 Society of Chemical Industry.

This study develops a pH‐responsive vanillin‐Schiff base microcapsule for controlled release of pyraclostrobin. It enables targeted release triggered by the fungal microenvironment, exhibiting superior UV resistance, adhesion, antifungal efficacy, and biocompatibility, offering a green strategy for intelligent crop disease control.

1. INTRODUCTION

Global population growth continues to drive up demand for food quantity and quality. However, climate change and increasingly severe pest and disease outbreaks cause ≤40% of food crop losses, which presents a grave challenge to global food security. ¹ Currently, chemical pesticides remain the primary strategy for crop pest and disease management, ² but their actual utilization efficiency is constrained by several drawbacks, including off‐target drift from crop surfaces, photodegradation sensitivity, and susceptibility to microbial metabolism, hydrolysis and photolysis in the environment. ³ , ⁴ Notably, 70% of active ingredients may be lost via droplet drift and run‐off during foliar application. ⁵ , ⁶ This not only significantly reduces pesticide application efficiency, but also leads to substantial environmental accumulation, endangering ecosystem stability and human health. ⁴ Consequently, developing green, efficient and sustainable pesticide delivery technologies has emerged as an urgent and critical challenge in modern agriculture.

Against this backdrop, sustained‐release pesticide formulations offer significant advantages by maintaining active ingredient concentrations over extended periods and enhancing disease control efficacy. Encapsulating pesticide active ingredients within controlled‐release carriers to achieve targeted and sustained release represents a key strategy for improving utilization efficiency. ⁷ Common carriers include liposomes, polymeric micelles, nanomaterials and microcapsules. ⁸ , ⁹ , ¹⁰ , ¹¹ , ¹² Among these, microcapsules have attracted widespread attention owing to their core‐shell structure, which effectively isolates the active ingredient from the external environment, reduces photolysis and degradation rates, lowers toxicity to nontarget organisms and prolongs effective duration. ¹³ , ¹⁴ Various preparation methods are available, including interfacial polymerization, solvent evaporation and in situ polymerization. ¹⁵ , ¹⁶ , ¹⁷ , ¹⁸ , ¹⁹ Interfacial polymerization has become the mainstream process owing to its simple operation, rapid encapsulation, mild conditions and high drug‐loading capacity, and favorable industrialization potential. ²⁰ , ²¹ , ²² Therefore, development efficient and stable microencapsulation technologies is of great significance for promoting pesticide reduction and efficiency enhancement, as well as advancing the transition to green agriculture advancing.

Currently, smart microcapsules with environment‐responsive release capabilities have become a leading research direction in pesticide formulation development. Among these, pH‐responsive systems hold broad application prospects, as they enable on‐demand release tailored to the acidic microenvironment associated with plant diseases. ²³ In recent years, Schiff bases, as one of the key bond types in dynamic covalent chemistry, have provided new strategies for designing intelligent functional materials. ²⁴ Schiff bases are produced through the condensation reaction of aldehydes/ketones with amine compounds. They contain a dynamic imine structure in their molecules and exhibit excellent pH responsiveness: reversible hydrolysis under acidic conditions and high stability under weakly alkaline conditions. This property gives them significant potential for use in the construction of targeted release systems, and they already have been widely applied in the fields of biomedicine and pesticides. ²⁵ However, pristine Schiff base‐based materials [e.g. small molecules and covalent organic frameworks (COFs)] are typically brittle and struggle to maintain structural integrity. By contrast, polyurethane possesses excellent toughness, elasticity, wear resistance and film‐forming properties, showing good application potential in pesticide adjuvants and thus making it an ideal candidate for modification. ²⁶ , ²⁷ , ²⁸ The raw materials for traditional polyurethanes (polyols and isocyanates) are primarily derived from petroleum, which imposes certain limitations on their application in pesticides. By contrast, the use of natural products and their derivatives as raw materials for the preparation of microcapsules or surface coatings has attracted widespread attention. ²⁹ Among these, naturally derived vanillin can serve as an aldehyde group donor; its aromatic aldehyde structure not only forms stable Schiff bases with amines, but also possesses multifunctional properties including UV resistance, antioxidant properties and antibacterial activity, making it highly suitable for the design of agricultural formulations. ³⁰ , ³¹ Furthermore, the rigid benzene ring in vanillin can significantly enhance the mechanical strength, thermal stability and chemical resistance of polyurethane backbones. This effectively overcomes the limitations of traditional polyurethanes namely, insufficient mechanical properties and poor high‐temperature resistance, while simultaneously serving the dual purposes of utilizing green raw materials and functional modification. Therefore, vanillin‐modified polyurethane represents an ideal raw material system for preparing pH‐responsive microcapsules with dynamic Schiff‐base linkages.

Pyraclostrobin (Pyr) is a broad‐spectrum, highly effective methoxyacrylate fungicide that can effectively control >100 plant diseases, such as powdery mildew, downy mildew, anthracnose, leaf spot, and rust, caused by ascomycetes, basidiomycetes, deuteromycetes and oomycetes, and is effective on a wide range of crops. Nevertheless, its practical application is limited by poor light stability, short residual efficacy and relatively high toxicity to nontarget organisms. ³² , ³³ , ³⁴ , ³⁵ Therefore, in this study, a pH‐responsive sustained‐release microcapsule system of Pyr containing dynamic Schiff‐base covalent bonds was fabricated via interfacial polymerization combined with electrostatic self‐assembly. The prepared microcapsules were characterized in terms of morphology, encapsulation efficiency, and photostability. Their release behavior under different pH conditions, as well as wetting and adhesion properties on rice leaf surfaces, were investigated systematically. Furthermore, the antifungal effects on various plant pathogens, including Rhizoctonia solani, Phomopsis sp., Botryosphaeria dothidea, Trichoderma spp., Mucor spp. and Fusarium spp. were examined, accompanied by assessments of acute toxicity to zebrafish and impacts seed germination. Experimental results indicate that the microcapsules possess good pH‐responsive release behavior, excellent light stability and leaf‐surface spreading ability, significant fungicidal activity and low environmental toxicity. This work provides a theoretical foundation for the development of a new generation of green and smart pesticide formulations.

2. MATERIALS AND METHODS

2.1. Materials

Vanillin (VAN; 98%), polyvinyl alcohol (PVA1788), diphenylmethane diisocyanate (MDI; 90%), triethylenetetramine (TETA; 70%) and n‐butyl acetate AR (99.0%) were obtained from Aladdin Reagent (Shanghai) Co., Ltd (China). EL‐40 was acquired from the Haian Petrochemical Plant (Jiangsu, China). Methanol (≥99.9%) was purchased from Chengdu Jinshan Chemical Reagents Co., Ltd (China). Pyraclostrobin (Pyr; 98%) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd (China).

2.2. Synthesis of vanilla based Schiff Base (VT)

Synthesis used the method described previously ³⁶ with the following modifications: 1.54 g (0.01 mol) TETA was placed into a 50‐mL round‐bottom flask. Subsequently, 2.07 g (0.01 mol) VAN was solubilized in ethanol (10 mL). The VAN liquid solution was then added dropwise to the round‐bottom flask via a funnel at room temperature (RT), and the reaction was permitted to continue for 10 h. During the reaction, the color gradually changed from colorless to orange, and finally to burgundy. The mixture was subjected to concentration at 60 °C, and the target product was obtained after freeze‐drying.

2.3. Synthesis of Pyr@PU microcapsules

A 2% PVA aqueous solution was prepared first. 17.115 g of this PVA solution were placed in a beaker 0.36 g EL‐40 was added and the mixture was stirred until completely dissolved to form the aqueous phase. Next 4.5 g Pyr and 6.9 g n‐butyl acetate were added into a beaker, and the mixture was heated to dissolve, cooled to RT after complete dissolution, and then 1.125 g MDI was added to form the oil phase. In an ice bath, the oil phase was slowly added dropwise to the aqueous phase under shearing at 8000 rpm. High‐speed shearing was continued for 10 min to obtain a stable emulsion. The 20 mL VT aqueous solution (1.55 wt%) were slowly added to a beaker equipped with a mechanical stirrer. The emulsion was subsequently heated to 40–50 °C and kept at this temperature for 2–3 h to yield the final product. The product was then harvested and washed thrtee times with deionized water and one to two times with a 1: 1 ethanol‐water solution, and then freeze‐dried for 48 h.

2.4. Calculation of encapsulation efficiency (EE) and loading capacity (LC)

The EE and LC of Pyr@PU microcapsules loaded with Pyr were determined by UV spectrophotometry. A series of Pyr@PU aqueous‐methanol solutions with concentrations of 5, 10, 15, 20 and 25 mg L⁻¹ were prepared, and their absorbance (A) was analyzed at 275 nm. A standard calibration curve (y = 0.0173x + 0.001, R ² = 0.9961) was established to calculate the EE and LC of the microcapsules. ³⁷

For sample determination, 0.010 g Pyr@PU microcapsules loaded with Pyr was added to a methanol–water solution (100 mL, v/v = 1/1), followed by sonication to achieve complete dissolution. Following centrifugation at 4000 rpm for 5 min, the supernatant was collected and analyzed at λ = 275 nm. The EE and LC were then calculated based on the above standard curve.

$$\mathit{EE}\%=\frac{C_{0}V}{W_0}\times 100\%$$

$$\mathit{LC}\%=\frac{C_{0}V}{W}\times 100\%$$

Where C ₀ is the concentration of Pyr@PU microcapsules, V is the total volume of the solution, W ₀ is the mass of the weighed sample and W is the initial amount of the weighed pesticide.

2.5. Characterization

The surface morphology of the Pyr@PU microcapsules was recorded using a scanning electron microscope (SEM S‐4800; Hitachi, Tokyo, Japan). Analysis was carried out using a Fourier transform infrared (FTIR) spectrometer (Spectrum Two; PerkinElmer, Shelton, CT, USA). The average particle size of the Pyr@PU microcapsules was monitored by dynamic light scattering (DLS) using a nanoparticle size analyzer (NS‐90 Z; Malvern Panalytical, Shanghai, China). Thermal gravimetric analysis (TGA) was performed on a STA 6000 instrument (PerkinElmer) under a 10 mL min⁻¹ N₂ flow, heated at 10 °C min⁻¹ from 0 to 800 °C.

2.6. In vitro controlled release kinetics

In order to investigate the pH‐responsive release behavior of Pyr@PU microcapsules, in vitro release tests under different pH conditions were carried out via the dialysis method. A 30mg sample was weighed and dispersed in a dialysis bag (3500 Da), which was then soaked in a mixed methanol–water solution (90 mL, v/v = 3/7) at pH 4.0, 7.0 and 9.0 respectively. The system was maintained at 25 °C with continuous stirring at 150 rpm. At specific time intervals, the release medium (4 mL) was collected into a test tube, and an equal volume of fresh dialysis buffer was supplemented to the system. The absorbance was measured at λ = 275 nm by UV spectrophotometry, and the cumulative release mass (m) was calculated from the standard curve according to the following formula.

$$m=\left(C_t+\frac{v}{V}\sum _0^{t-1}{C}_t\right)\times V$$

where C _t is the Pyr concentration at time t, v is the sampling volume (4 mL) and V is the total release volume (90 mL).

Additionally, to investigate the release kinetics under different pH conditions, the Pyr release profiles from Pyr@PU microcapsules were fitted using zero‐order, first‐order, Higuchi and Ritger–Peppas models via the following equations.

$$\mathrm{\text{Zero}}-\mathrm{\text{order model}}:{\mathsf{M}}_{\mathsf{t}}/{\mathsf{M}}_{\mathsf{z}}=\mathsf{kt}$$

$$\mathrm{\text{First}}-\mathrm{\text{order model}}:{\mathsf{M}}_{\mathsf{t}}/{\mathsf{M}}_{\mathsf{z}}=\mathrm{1}-{\mathrm{e}}^{-\mathsf{kt}}$$

$$\mathrm{\text{Higuchi model}}:{\mathsf{M}}_{\mathsf{t}}/{\mathsf{M}}_{\mathsf{z}}={\mathsf{kt}}^{\mathrm{0.5}}$$

$$\mathrm{\text{Ritger}}–\mathrm{\text{Peppas model}}:{\mathsf{M}}_{\mathsf{t}}/{\mathsf{M}}_{\mathsf{z}}={\mathsf{kt}}^{\mathsf{n}}$$

M _t /M _z is the cumulative Pyr released fraction at times t (Mz = total encapsulated Pyr mass), k is the kinetic constant and n is the diffusion exponent. For spherical samples, n ≤ 0.45 (Fickian diffusion), 0.45 < n < 0.89 (non‐Fickian) and n ≥ 0.89 (zero‐order release). ³⁸

2.7. Study on the UV‐protective properties of Pyr@PU microcapsules

In order to evaluate the UV resistance of the Pyr@PU microcapsule samples, the experiment was tested by optimizing the method described by Luo et al. ³⁹ Typically, ≈30 mg Pyr@PU microcapsules were dispersed in a dialysis bag with a molecular weight cutoff of 3500 Da and submerged in of a methanol/water mixture (90 mL, v/v = 3/7). The system was then exposed to irradiation from an 8 W germicidal UV lamp (λ = 254 nm, L = 15 cm, 25 °C). During the irradiation period, 4 ‐mL aliquots of the release medium were withdrawn at set time intervals, and fresh buffer medium (4 mL) was supplemented promptly to preserve a constant volume. The concentration of released Pyr was determined by UV spectrophotometry using Pyr as the standard reference. All experiments were repeated three times. The corresponding calculation formula is as follows:

$$\text{Cumulative photolyses proportion}\left(\%\right)=\left(C_t+\frac{v}{V}\sum _0^{t-1}{C}_t\right)\times V$$

Wher C _t is the Pyr concentration at time t, v is the sampling volume (4 mL) and V is the total release volume (90 mL).

2.8. Bioactivity studies

2.8.1. In vitro antifungal activity

The in vitro antifungal effects of Pyr and Pyr@PU microcapsules against R. solani, Phomopsis sp., B. dothidea, Trichoderma spp., Mucor spp. and Fusarium spp. were assessed using a mycelial growth inhibition test. Sample solutions with a series of concentrations were incorporated into molten PDA solutions and uniformly spread on agar plates. After solidification, a mycelial plug was positioned in the middle of each plate and cultured at 25 °C for 5 days. The diameter of mycelial colonies was evaluated via the cross‐counting method, and the antifungal effects was calculated according to the corresponding formula. All experiments were performed in triplicate. ⁴⁰

$$\mathit{\text{Inhibition rate}}\left(\%\right)=\frac{\mathit{CK}-C_1}{\mathit{CK}-0.5}\times 100\%$$

where CK is the blank control mycelium diameter (cm), C ₁ is the treated mycelial diameter (cm) and 0.5 is the inoculum disc diameter (0.5 cm).

2.8.2. In vivo antifungal activity

Protective and therapeutic efficacies of Pyr@PU microcapsules against R. solani (cause of rice sheath blight) and Phomopsis sp. (cause of stem rot) were evaluated using technical‐grade Pyr (positive control) and a 0.1% Tween‐80 aqueous mixture (blank control). ⁴¹ Test suspensions were prepared by dispersing Pyr or Pyr@PU microcapsules at 200 μg mL⁻¹ in 0.1% Tween‐80 aqueous mixture. Healthy rice leaves and kiwifruit of uniform size were disinfected with 75% ethanol followed by sterile water, and blotted dry with filter paper. The rice sheath blight fungus and the stem spot fungus were cultured 1 week before the antifungal assay.

Samples were uniformly sprayed with the 200 μg mL⁻¹ test solution until run‐off, with 0.1% Tween‐80 serving as the control. After incubating in an incubator at 25 °C for 24 h, the leaves and fruits were inoculated at the center (rice) and middle (kiwifruit) with mycelial plugs of the respective pathogens. After 5–7 days of incubation, the lesion diameters were recorded and determined; with each treatment was repeated in triplicate.

Rice leaves and kiwifruit were first inoculated with mycelial plugs of R. solani and Phomopsis sp., respectively, and allowed to incubate in an incubator (24 h). Subsequently, the samples were sprayed with the 200 μg mL⁻¹ test solution or 0.1% Tween 80 (control) until thoroughly moistened. After 5–7 days of incubation, the size of the lesions was examined and determined; with each treatment was repeated in triplicate.

Use the following formula to determine the efficacy of prevention and treatment:

$$P\mathit{\text{or}}C\left(\%\right)=\frac{D_{\mathrm{c}}-D_{\mathrm{t}}}{D_{\mathrm{c}}-1}\times 100\%$$

where P and C are the protective and therapeutic effects, respectively, D _t is the lesion diameter in protective or therapeutic group (cm), D _c is the lesion diameter in blank control group (cm) and 1 is the fungal stem diameter (cm).

2.9. Safety assay

2.9.1. Acute toxicity in zebrafish

The acute toxicity of Pyr@PU microcapsules was evaluated according to OECD 203 and the Chinese National Standard GB/T 31270.12–2014 for Environmental Safety Assessment of Chemical Pesticides. ⁴² A static acute toxicity test was performed using zebrafish (Danio rerio). with nine test concentrations were set in a preliminary experiment, with each aquarium containing test solution (3 L) and a blank control group established in parallel. Each tank was stocked with 10 healthy zebrafish of uniform size, with triplicate setups per group. Zebrafish survival was assessed at 24, 48, 72 and 96 h; Zebrafish were considered dead when showing no reaction to mechanical stimulation. No food was provided during the entire test period. In environmental safety assessments, the toxicity classification of pesticides to zebrafish was classified according to the median lethal concentration (LC₅₀) as follows: extremely toxic (LC₅₀ ≤ 0.1 mg L⁻¹); highly toxic (0.1 < LC₅₀ ≤ 1.0 mg L⁻¹); moderately toxic (1.0 < LC₅₀ ≤ 10.0 mg L⁻¹) and low toxicity (LC₅₀ > 10.0 mg L⁻¹).

2.9.2. Seed germination experiment

The phytotoxicity was evaluated by a rice seed germination assay, following a modified previous method. ⁴³ Thirty rice seeds were soaked in pesticide solutions of varying concentrations for 12 h. Filter paper of the same diameter was placed in a 9‐cm‐diameter petri dish as a germination medium, and moistened with an appropriate amount of deionized water. The pretreated seeds were then evenly arranged on the filter paper and covered with a dish lid. All samples were incubated in an incubator at 20–25 °C, with distilled water set as the blank control (CK). germination was observed after several days of continuous incubation. To ensure optimal germination conditions, monitor the germination status and moisture levels of the seeds daily during the germination period, and replenish water as necessary.

$$\mathrm{\text{Germination rate}}=\begin{array}{l}\left(\mathrm{\text{Number of germinated seeds}}/\mathrm{\text{Total seeds}}\right)\\ \times \mathrm{100}\%\end{array}$$

3. RESULTS AND DISCUSSION

3.1. Synthesis and characterization of microcapsules

Schiff bases are acid‐responsive structures formed by the condensation of amine compounds with active carbonyl compounds; they readily dissociate under acidic conditions and are therefore widely used in the preparation of acid‐responsive controlled‐release materials. In this study, the natural organic compounds VAN and TETA were used as starting materials to synthesize an acid‐responsive smart precursor material, denoted VT, as shown in Fig. 1(A). A slight excess of TETA was used to ensure the complete reaction of VAN. An acid‐sensitive VT material with a Schiff base structure was successfully synthesized at 25 °C with a high yield (71.65%). Visually, VT is an orange‐yellow compound [Fig. 1(B)] with low solubility in ethanol and high solubility in water.

Figure 1.

(A) Synthesis of Schiff base. (B) Morphology of VT and its solubility in different solvents. (C) Synthesis route of Pyr@PU microcapsules.

Nano‐microcapsules loaded with the fungicide Pyr (Pyr@PU) were prepared by interfacial polymerization [Fig. 1(C)], with LC of 80.89%and EE of 96.17%. During emulsification, isocyanate droplets containing the fungicide Pyr underwent cross‐linking via interfacial polymerization in an ice bath, where –NCO groups in the droplets reacted with aqueous‐phase vanillyl triamine. This gradual curing at the water–oil interface formed a microcapsule shell structure, thereby encapsulating the fungicide‐loaded isocyanate within the core. Observation of the microcapsule core‐shell structure using an optical microscope [Fig. 2(A)] clearly revealed the core‐shell morphology of the microcapsules. Because the isocyanate was encapsulated within the microcapsule core, and the excess highly reactive –NCO groups in the system could fully react with the vanillin‐Schiff base structure, thereby minimizing potential safety risks associated with isocyanates toxicity. Additionally, microcapsules were synthesized via interfacial synthesis, which involves only water treatment. Upon release, the highly reactive isocyanates rapidly react with atmospheric moisture and active hydrogen (e.g. hydroxyl groups) in the matrix. The resulting polyurethane and polyurea exert no harm to human health or the environment. ⁴⁴

Figure 2.

(A) Optical micrograph; (B) SEM image; (C) particle size distribution; (D) FTIR spectra; (E) TGA curve of Pyr@PU microcapsules.

This reaction proceeded to completion, and excess isocyanate was encapsulated within the microcapsule core via interfacial polymerization, thus avoiding the waste of vanillin, Schiff base and isocyanate. Consequently, the microcapsule synthesis process facilitates efficient resources utilization and environmental sustainability. Imaging by SEM [Fig. 2(B)] revealed that Pyr@PU exhibits a spherical morphology, with the microspheres displaying a relatively uniform spherical shape, thereby confirming the successful preparation of the microspheres. The particle size distribution of microcapsules is shown in Fig. 2(C), the average particle size is 459 nm with a polydispersity index (PDI) of 0.318, indicating a narrow size distribution and favorable dimensional uniformity. Such uniform particle dimension benefits the suspension stability of the resulting microcapsules. FTIR analysis showed that Pyr@PU microcapsules had been successfully synthesized. As shown in Fig. 2(D), The following Pyr@PU characteristic peaks can be seen in the spectrum: 2954 cm⁻¹ (methyl stretching vibration), ⁴⁵ 1500 cm⁻¹ (aromatic ring C=C) ⁴⁶ and a broad absorption peak belonging to the N—H bond of the amino ester in the range of 3000–3500 cm⁻¹, ⁴⁷ indicating the formation of amino ester bonds in the crosslinking reaction. In the high wavenumber region (3400–5000 cm⁻¹) of the spectrum, pure Pyr exhibits strong absorption at 3400–3700 cm⁻¹ owing to O—H and N—H stretching vibrations, implying the presence of strong hydrogen (H)‐bonding interactions. By contrast, in Pyr@PU the transmittance significantly increases and the absorption significantly decreases. This change is attributed to the effective shielding of the polar groups (‐OH, ‐NH₂) in Pyr by the PU shell, which may have disrupted its original H‐bonding network, confirming the successful encapsulation of Pyr inside the PU shell. Additionally, a weak absorption peak was observed, corresponding to unreacted isocyanate groups (‐NCO), ⁴⁸ further indicating that isocyanates are encapsulated within the microcapsule shell. The thermal stability of Pyr@PU microcapsules and technical‐grade Pyr was evaluated by TGA [Fig. 2(E)]. Both samples exhibited a multi‐step weight‐loss profile as temperature rose. The weight loss of Pyr@PU and Pyr occurred in two main stages, corresponding to the thermal decomposition of Pyr (250–450 °C) and further breakdown of the resulting products (460–800 °C). The degradation curves of Pyr@PU and Pyr closely overlapped in the range of 250–330 °C, providing additional evidence that Pyr was successfully encapsulated within the Pyr@PU microcapsules.

3.2. Analysis of the in vitro release properties of Pyr@PU microcapsules

Given that a Schiff base is unstable under acidic conditions, a release kinetics analysis was conducted on Pyr@PU microcapsules. ⁴⁹ The release behavior of microcapsules under different pH environments is shown in Fig. 3(A). Sustained release was observed over 144 h at pH 4.0, 7.0 and 9.0, with cumulative release rates of 82.81%, 63.50% and 70.33%, respectively. The highest release efficiency was achieved at pH 4.0, which was 19.31% and 12.48% higher than those under neutral and alkaline conditions. The accelerated drug release under acidic environments originates from two synergistic effects of the microcapsule shell. On the one hand, acid‐triggered cleavage of dynamic Schiff‐base covalent bonds gradually breaks the crosslinked shell framework; on the other, protonation of shell functional groups induces polymer swelling and generates abundant penetrable pore channels inside the shell. ⁵⁰ Combined structural disintegration and pore formation collectively facilitate the continuous outflow of pyraclostrobin from Pyr@PU microcapsules. Additionally, the biological activity against R. solani in media of varying pH [Fig. 3(B)] showed that the inhibition rate of the Pyr@PU group reached 65.09 ± 1.82% at pH 4.0, significantly higher thanat pH 7.0 (43.27 ± 1.50%) and pH 9.0 (44.73 ± 1.30%). These results further confirm that the microcapsules have acid responsive release characteristics. Furthermore, we found that the release rate under alkaline conditions was higher than under neutral condition, which may be attributed to the increased porosity of the microcapsule shell under alkaline conditions, the base‐catalyzed exchange reaction of free amines, as well as non‐pH‐related factors such as the composition of the buffer solution or ionic strength. ⁵¹ This phenomenon indicates that the design of pH‐responsive delivery systems should not only focus on the kinetics of chemical bond cleavage, but also must comprehensively consider changes in the physical structure of the carrier material and the overall response behavior of the system.

Figure 3.

(A) Controlled‐release kinetics of Pyr@PU microcapsules at pH 4.0, 7.0 and 9.0. (B) Inhibitory effects of Pyr@PU microcapsules on R. solani at pH 4.0, 7.0 and 9.0. (C) Fitting models of Pyr@PU microcapsules at pH 4.0, 7.0 and 9.0.

In order to further investigate Pyr from Pyr@PU microcapsules, the release data were fitted with four kinetic models [Fig. 3(C) and Table 1]. The correlation coefficients revealed that the release behavior of Pyr was optimally described by the first‐order model at pH 4.0. By contrast, the release profiles at pH 7.0 and 9.0 fitted better with the Ritger–Peppas model, with the diffusion exponent n calculated as 0.38 and 0.38, respectively. All obtained n values were <0.45, confirming that the drug release from Pyr@PU microcapsules obeys typical Fickian diffusion mechanism. ⁵²

Table 1.

Fitting models of Pyr@PU microcapsules at pH 4.0, 7.0 and 9.0

Model	Slow‐release environment	K	R 2	n
Zero‐order	pH = 4.0	0.5081	0.7649	‐
	pH = 7.0	0.3891	0.8197	‐
	pH = 9.0	0.4505	0.8107	‐
First‐order	pH = 4.0	0.0433	0.9917	‐
	pH = 7.0	0.0502	0.9267	‐
	pH = 9.0	0.0556	0.9197	‐
Higuchi	pH = 4.0	7.2330	0.9241	‐
	pH = 7.0	5.2985	0.9540	‐
	pH = 9.0	5.9820	0.9463	‐
Ritger–Peppas	pH = 4.0	13.0050	0.9478	0.39
	pH = 7.0	9.9692	0.9765	0.38
	pH = 9.0	11.1004	0.9699	0.38

3.3. Study on the UV‐protective properties of Pyr@PU microcapsules

Because Pyr is highly sensitive to light, which is unstable and prone to decomposition under UV irradiation, the UV‐resistant properties of Pyr@PU microcapsules were systematically investigated. As shown in Fig. 4(A), after 60 h of irradiation with an UV lamp (8 W; 254 nm), the degradation rate of Pyr was 92%, whereas that of Pyr@PU microcapsules was only 68%, significantly lower than that of Pyr. These results indicate that encapsulating Pyr using nanomaterials can enhance its photostability. This is attributed to the Schiff‐base bond, which not only encapsulates Pyr within the core to achieve acid‐responsive controlled release, but also blocks light, thereby reducing Pyr degradation. ⁵³ Furthermore, the antifungal activities against R. solani were evaluated after formulating Pyr@PU microcapsules and technical‐grade Pyr into 2 μg mL⁻¹ solutions and exposing them to UV irradiation for 10 h [Fig. 4(B)]. The inhibition rate of microcapsules is 26.56 ± 1.39%, which was 9.68% higher than that of pure Pyr (16.88 ± 1.02%). Therefore, nano‐encapsulation of the drug significantly improves the photostability and utilization efficiency of Pyr, achieving synergistic effects while reducing pesticide dosage. This accordingly lowers pesticide residue levels and renders the formulation more environmentally friendly.

Figure 4.

(A) UV resistance of Pyr@PU microcapsules and technical‐grade Pyr. (B) Inhibitory activity of Pyr@PU microcapsules and technical‐grade Pyr after dark storage and UV irradiation for 10 h.

3.4. Wetting and adhesion properties of Pyr@PU microcapsules on Rice leaves

The wetting and adhesion properties of pesticides on leaf surfaces directly determine their deposition and utilization efficiency. Owing to the highly hydrophobic nature of rice leaf surfaces, the spray solution tends to run off after application, making it difficult for the pesticide to exert its efficacy and resulting in low pesticide utilization rates. Therefore, it is necessary to investigate their wettability and adhesion on rice leaves. As shown in Fig. 5(A), after simulated rain wash‐off, the retention rates of Pyr@PU microcapsules and Pyr on rice leaves were 48.70% and 39.10%, respectively, indicating that Pyr@PU microcapsules displayed stronger resistance to rain wash‐off on rice leaves compared with Pyr. This may be attributed to the presence of numerous polyphenolic groups on the surface of the Pyr@PU microcapsules, which allow these groups to form H‐bonds with the leaf surface, thereby markedly enhancing adhesion to the leaf surface. ⁵⁴ As shown in Fig. 5(B), pure water barely spreads on rice leaves, with the contact angle remaining at 135 ± 0.4°, indicating that the leaves are superhydrophobic. Pyr@PU microcapsules exhibited better spreading behavior, with a contact angle of 116°± 0.3°. This is attributed to the polyphenol structures within the Pyr@PU microcapsules, which significantly improve spreading. ⁵⁵ In summary, Pyr@PU microcapsules promote the deposition and spreading of active ingredients on rice leaves.

Figure 5.

(A) Adhesion efficiency of Pyr and Pyr@PU microcapsules on rice leaf surfaces. (B) Dynamic contact angle changes of water and Pyr@PU microcapsules suspensions on rice leaves.

3.5. Bioactivity testing of Pyr@PU microcapsules

Mycelial growth rate assay was used to evaluate the in vitro antifungal activity of Pyr and Pyr@PU against R. solani, Phomopsis sp., B. dothidea, Trichoderma spp., Mucor spp. and Fusarium spp. The results showed that both Pyr and Pyr@PU exhibited concentration‐dependent inhibitory effects against the aforementioned six pathogens, and the corresponding half‐maximal effective concentrations (EC₅₀), potency regression equations and correlation coefficients. As shown in Table 2, the inhibitory activity of Pyr@PU microcapsules against six pathogens most of was higher than or equivalent to that of Pyr. Specifically, The EC₅₀ value of Pyr@PU against R. solani was 0.1878 μg mL⁻¹, which was 2.9‐fold more active than Pyr (0.5456 μg mL⁻¹). Against Phomopsis sp., the EC₅₀ value of Pyr@PU was 0.3534 μg mL⁻¹, with an activity 2.0‐fold that of Pyr (0.6923 μg mL⁻¹) [Fig. 6(A), (B)]. These results indicate that encapsulating Pyr in nanomaterials not only enables pH‐responsive controlled release, but also significantly enhances its antibacterial activity. Additionally, Pyr@PU exhibits slightly weaker short‐term activity against B. dothidea  than technical Pyr. This arises because the microcapsule shell restrains initial burst release and reduces instantaneous drug concentration around hyphae relative to free technical Pyr. Species‐specific differences in fungal cell wall structure and permeability lead to varied susceptibility to slow‐release formulations, explaining the slightly lower in vitro efficacy against B. dothidea.

Table 2.

EC₅₀ curves for Pyr@PU microcapsules and Pyr against six fungal species

Strain	Samples	EC50(μg mL−1)	R2	Equation (y=)
R. solani	Pyr	0.5456	0.9960	0.9242x + 5.2432
	Pyr@PU	0.1878	0.9803	0.7652x + 5.5557
Phomopsis sp.	Pyr	0.6923	0.9893	0.7137x + 5.1140
	Pyr@PU	0.3534	0.9988	0.6345x + 5.2866
B. dothidea	Pyr	0.4488	0.9791	0.9445x + 5.3286
	Pyr@PU	0.5023	0.9886	1.1189x + 5.3346
Trichoderma spp.	Pyr	1.4889	0.9749	0.8544x + 4.8523
	Pyr@PU	1.3693	0.9925	0.9788x + 4.8664
Mucor spp.	Pyr	2.0774	0.9845	0.7020x + 4.7771
	Pyr@PU	1.9556	0.9906	0.6348x + 4.8151
Fusarium spp.	Pyr	0.6346	0.9810	0.7326x + 5.1447
	Pyr@PU	0.5044	0.9852	0.8297x + 5.2466

Figure 6.

(A) EC₅₀ curves of Pyr@PU microcapsules and technical‐grade Pyr against R. solani. (B) EC₅₀ curves of Pyr@PU microcapsules and technical‐grade Pyr against Phomopsis sp. (C, D) Curative and protective inhibition rates of Pyr@PU microcapsules and technical‐grade Pyr on rice leaves and kiwifruit at 200 μg mL⁻¹. (E, F) Corresponding photographs of protective and curative effects at the same concentration. Significance: *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus technical‐grade Pyr.

In order to thoroughly validate the biological activity of Pyr@PU, its protective and curative effects were further evaluated on isolated rice leaves and kiwifruit. As shown in Fig. 6(C), (D), at 200 μg mL⁻¹, Pyr@PU demonstrated superior protective and curative effects compared to Pyr in both systems, with the protective effects generally outperforming the curative effects. As illustrated in Fig. 6(E), on rice leaves infected with R. solani, the protective and curative efficacies of Pyr@PU reached 57.90% and 37.89%, respectively, which were 4.93% and 2.38% higher than those of Pyr (52.97% and 35.51%), demonstrating a clear synergistic effect. In in vivo experiments on kiwifruit infected with Phomopsis sp. [Fig. 6(F)], the protective and curative efficacies of Pyr@PU were 79.62 ± 4.33% and 67.59 ± 3.86% respectively, which were markedly higher than 45.86 ± 2.78% and 28.28 ± 2.16% observed for Pyr, representing synergistic increases of 33.76% and 39.31%, respectively.

The synergistic mechanism of Pyr@PU can be attributed to two factors. first, the microcapsules respond to the acidic environment characteristic of the site of pathogen infection, causing the cleavage of Schief base bond and enabling the targeted controlled release of the pesticide, thus optimizing the utilization efficiency of the active ingredient. Second, the microcapsules possess excellent UV resistance and good adhesion to leaf surfaces. This minimizes unnecessary loss of the pesticide during the early stages of disease infection (the protective phase), allowing more active ingredients to remain and exert a long‐lasting antimicrobial effect. In summary, Pyr@PU demonstrated superior control efficacy compared to the traditional Pyr formulation on both rice leaf and kiwifruit systems, with protective effects consistently better than curative effects. These results indicate that such smart delivery systems hold significant necessity and immense application potential for proactive prevention and early control in agricultural production.

3.6. Biosafety assessment

3.6.1. Zebrafish experiments

With the widespread application of nanomaterials in agriculture, the biosafety of these materials has become a critical consideration. Given that Pyr exhibits high toxicity to aquatic organisms, using zebrafish as a model organism for in vivo toxicity evaluation is of great significance for evaluating the biosafety of pesticide formulations. Therefore, it is essential to conduct a biosafety evaluation of Pyr@PU microcapsules on aquatic organisms. As shown in Table 3, the LC₅₀ of technical‐grade Pyr to zebrafish within 96 h was 0.054 mg L⁻¹, indicating its high toxicity to aquatic organisms. By contrast, the LC₅₀ of Pyr@PU microcapsules to zebrafish was 0.122 mg L⁻¹ (R ² = 0.9819), which was 2.26‐fold higher than that of technical‐grade Pyr, demonstrating significantly reduced acute toxicity to aquatic organisms. The results of the acute toxicity test showed that the toxicity of Pyr@PU microcapsules to zebrafish was significantly lower than that of technical‐grade Pyr. This is mainly because the microcapsule structure can slow down the release rate of the active ingredient, reduce the concentration of the active ingredient in the water environment, and thus reduce the toxic effect on aquatic organisms. This finding further confirms that the nano‐encapsulation technology can effectively reduce the environmental risk of pesticides while ensuring the biological activity of the active ingredient, which is consistent with the concept of green pesticide development.

Table 3.

Toxicity assessment of Pyr@PU microcapsules and Pyr in zebrafish

Analytes	Exposure time (h)	LC50 (mg L−1)	95% confidence interval	R 2	Equation (y=)
Pyr@PU	24	0.156	0.143–0.394	0.9857	9.7249x + 12.853
	48	0.145	0.130–0.197	0.9890	9.6151x + 13.067
	72	0.137	0.086–0.170	0.9861	8.6967x + 12.499
	96	0.122	0.077–0.133	0.9819	12.114x + 16.041
Pyr	24	0.133	0.093–0.185	0.9448	3.0405x + 7.6867
	48	0.113	0.021–0.143	0.9982	3.6000x + 8.4101
	72	0.095	0.061–0.133	0.9022	2.8682x + 7.9682
	96	0.054	0.041–0.066	0.9914	5.2023x + 11.569

Note: n = 3.

3.6.2. Seed germination experiment

Under suitable conditions (e.g. moisture, temperature and oxygen), plant seeds can absorb water, swell and germinate. However, seed germination can be affected when exposed to certain drug concentrations. Therefore, assessing seed germination is an effective way to predict and evaluate the potential toxicity and bioavailability of drugs to plants. As shown in Fig. 7(A)–(C), compared with the blank control group different concentrations of Pyr@PU microcapsules and technical‐grade Pyr had no significant effect on rice seed germination. These results indicate that neither the Pyr@PU microcapsules nor the Pyr exerted a significant inhibitory influence on rice seed germination.

Figure 7.

Effects of Pyr@PU microcapsules (A) and Pyr (B) on germination rate and morphological status of rice seeds (C).

4. CONCLUSION

A pH‐responsive nanoparticle‐based pesticide delivery system (Pyr@PU) was developed. Utilizing a Schiff‐base structure, a new pH‐responsive microcapsule with a vanillin‐based Schiff‐base polyurethane shell and isocyanate core was successfully synthesized via interfacial polymerization. The obtained Pyr@PU microcapsules exhibited a particle size of 459 nm, with a Pyr loading capacity of 80.89%, and an encapsulation efficiency of 96.17%. Pyr@PU possessed superior UV stability, good leaf wettability and excellent rainfastness, as well as showed acid‐responsive sustained release to adapt to the acidic microenvironment after plant infection. Bioactivity assays verified that Pyr@PU microcapsules exerted prominent antifungal effects. At 200 μg mL⁻¹, Pyr@PU achieved protective and curative efficacies of 79.62% and 67.59% against kiwifruit pathogens, and 57.90% and 37.89% on rice leaves. Furthermore, biosafety evaluations using zebrafish and seed germination experiments demonstrated the biocompatibility and environmental safety of Pyr@PU microcapsules. This study offers a scalable route to eco‐friendly stimulus‐responsive controlled‐release materials and inspires the development of nanopesticides. The interfacial polymerization method also can be applied to prepare O/W nanomicelles for loading diverse pesticides and realizing targeted delivery.

CONFLICT OF INTEREST

The authors declare no conflicts of interest.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.