Neonicotinoid-based pesticides are extensively used owing to their broad insecticidal spectrum and activity. We developed neonicotinoid dinotefuran (DIN)-loaded chitosan-gelatin microspheres using a spray-drying technology, resulting in a pH- and temperature-responsive controlled-release system.
Carbohydrate Polymers 277 (2022) 118880 Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol Controlled release of dinotefuran with temperature/pH-responsive chitosan-gelatin microspheres to reduce leaching risk during application Qizhen Zhang, Yu Du, Manli Yu, Lirui Ren, Yongfei Guo, Qinghua Li, Mingming Yin, Xiaolong Li, Fuliang Chen * Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China A R T I C L E I N F O A B S T R A C T Keywords: Chitosan Gelatin Dinotefuran Microsphere Pesticide Trialeurodes vaporariorum Neonicotinoid-based pesticides are extensively used owing to their broad insecticidal spectrum and activity We developed neonicotinoid dinotefuran (DIN)-loaded chitosan-gelatin microspheres using a spray-drying technol ogy, resulting in a pH- and temperature-responsive controlled-release system Upon introducing chitosan into the triple-helix structure of gelatin, the physically modified gelatin microspheres became smooth, round, and solid, improving their thermal storage stability The spray-drying parameters were optimized using three-dimensional surface plots When scaled up under optimal conditions, the corresponding loading content and encapsulation efficiency were 21.5% and 98.17%, respectively Compared with commercial dinotefuran granules, our biode gradable composite carriers achieved the immobilization of dinotefuran to reduce pesticide leaching by 5.57–19.89% in soil, improved the soil half-life of DIN, and improved its cumulative absorption by plants Therefore, the microspheres showed better efficacy against Trialeurodes vaporariorum Our results confirm that this simple approach can improve the utilization efficiency of neonicotinoids, decrease leaching loss, and pro mote ecological safety Introduction Neonicotinoids are widely used because of their strong systemic properties and high insecticidal activity However, foliar neonicotinoid spraying is associated with the drift of pesticides, loss of liquid, and high toxicity to honeybees (Hatfield et al., 2021; Tsvetkov et al., 2017), causing environmental pollution and damaging the growers' economic interests (Li et al., 2019; Xiang et al., 2014) Although root application via seed treatment, hole/spot application, or root/drip irrigation may minimize such damage (Alford & Krupke, 2019), the high water solu bility of neonicotinoid readily leads to groundwater pollution following direct soil application (Berens et al., 2021), highlighting the potential of neonicotinoids to contribute to environmental loading Alford and Krupke (2019) indicated that neonicotinoid clothianidin, a seed treat ment for corn and soybeans, has been linked to waterway and irrigation water contamination Specific concentrations of neonicotinoids were detected in surface, ground-, and drinking water (Alford & Krupke, 2019); this contamination was considered to be a direct result of runoff and/or leaching In 1991, the United States Environmental Protection Agency (Farland, 1991) recommended a pollutant ecological risk assessment Hence, there is an urgent need to address the environmental pollution caused by neonicotinoid leakage Efforts to reduce pesticide loss and improve soil quality include adding activated carbon, humic acid, and peat to soil (Xie et al., 2017), thereby significantly reducing the amount of neonicotinoid leaching in the soil and protecting groundwater; Dai et al (2013) used biochar to treat the adsorption of atrazine (herbicide) to the soil However, in actual soil treatment, the high cost of adding materials, cumbersome operation, and difficulties in regeneration limit the widespread appli cation Alternatively, changing pesticide formulations to offer a controlled release may constitute a feasible and effective strategy to control leaching Imidacloprid granules (Yuan et al., 2020) and clo thianidin granules (Zhang et al., 2015) alleviate the low pesticide usage rate associated with spraying; however, leaching has not been evaluated and such systems generally only provide simple dissolution-regulated pesticide release, rendering it difficult to achieve controlled release in complex environments (Xiang et al., 2020) Consequently, the prepa ration of controlled release systems based on biodegradable carriers is attracting attention to enhance controlled-release performance Chitosan (CS) and gelatin (GEL) are widely used as microsphere * Corresponding author at: Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China E-mail address: chenful2005@aliyun.com (F Chen) https://doi.org/10.1016/j.carbpol.2021.118880 Received 25 June 2021; Received in revised form November 2021; Accepted November 2021 Available online 13 November 2021 0144-8617/© 2021 The Authors Published by Elsevier Ltd This is an open (http://creativecommons.org/licenses/by-nc-nd/4.0/) access article under the CC BY-NC-ND license Q Zhang et al Carbohydrate Polymers 277 (2022) 118880 composite carriers in medicine, owing to their biodegradability, low cost, and easy availability (Jalaja et al., 2016; Nagahama et al., 2009) The GEL polymer chain contains active amino, hydroxyl, and carboxyl groups that can bond to the reactive amino group of CS through the carboxyl group (Ma et al., 2020), with CS–GEL mixtures at appropriate ratios affording good overall performance (Wang et al., 2018) through the formation of flexible, stable polymer chains GEL crosslinks with CS to form a tight structure with temperature and pH sensitivity, func tioning as an “on–off” switch to regulate the release of cargo molecules from the “reservoir” (Xiang et al., 2020) Although numerous studies have evaluated the agricultural application of microsphere delivery systems (Grillo et al., 2011; Zhang et al., 2019), to the best of our knowledge, microspheres based on biodegradable CS-GEL to reduce dinotefuran leaching risk have not been reported Cucumber (Cucumis sativus L.), an economically essential vegetable in China, is prone to infestation by pests such as Trialeurodes vapor ariorum and aphids; thus, the use of chemical insecticides to control pests represents an important strategy to increase production In this study, we used the neonicotinoid insecticide dinotefuran (DIN) as a model pesticide to explore the feasibility of CS-physically modified GEL microspheres (DIN@CS-GEL) as a controlled-release carrier Spraydrying was used for simple and rapid encapsulation and the parame ters were optimized using three-dimensional (3D) surface plots The effects of different pH and temperature values on the intelligent release of DIN were determined in vitro The risk of groundwater contamination by the microspheres was assessed using soil leaching experiments DIN degradation in the soil, cumulative absorption in cucumber leaves and fruits, and control efficacy toward T vaporariorum were evaluated The findings provide a promising strategy for simple, low-cost, and intelli gent controlled release to decrease groundwater contamination risk potential and reduce the use of pesticides for sustainable plant protection water was used for all experiments 2.2 DIN-loaded CS-GEL microsphere preparation To prepare microspheres, CS (1.5%, w/v) was dissolved in doubledistilled water with acetic acid by stirring at room temperature (25 ± ◦ C) until clarified GEL solution (1.5%, w/v) was prepared by dis solving GEL in double-distilled water and stirring for 0.5 h at 50–70 ◦ C until reaching complete solubilization; then, we mixed the CS solution with the GEL solution to obtain a CS-GEL dispersion Thereafter, the DIN TC was completely dissolved in this dispersion The dispersion (500 mL) was nebulized through a nozzle (diameter 0.5 mm) using a spray-dryer (Labplant Basic Spray Dryer, North Yorkshire, England) in Fig 1A Microsphere preparation was optimized using Design Expert soft ware (Version 8.0.5, Stat-Ease Inc., USA) Pump rate (X1) and inlet temperature (X2) were selected as independent variables based on pre liminary studies, with loading content (Y) as the response variable To depict the interrelationship between independent and response vari ables, the obtained data and model were fitted using 3D surface plots (Zhang et al., 2019) 2.3 Sample characterization Fourier-transform infrared spectroscopy (FT-IR; Nicolet 6700, Thermo Scientific, Waltham, MA, USA) was used to determine whether the DIN was embedded into the DIN@CS-GEL, and FT-IR spectra were recorded (resolution cm− 1) Microsphere structural and morphological features were observed using images obtained on a SU8010 Ultra-HighResolution Scanning Electron Microscope (SEM, Hitachi, Tokyo, Japan) operated at an accelerating voltage DIN characteristics in microspheres were analyzed by derivative thermogravimetry (DTG), thermogravi metric analysis (TGA; TA Q600, TA Instruments, USA), and differential scanning calorimetry (DSC; DSC 4000, Perkin Elmer, Massachusetts, USA) Microsphere size and distribution were acquired using a laser particle size distribution analyzer (BT-2600, Baxter, Dandong, China) The mean particle size was measured following triplicate replication Xray photoelectron spectroscopy (XPS) was performed on a photoelectron spectrometer (Thermo Scientific K-Alpha, Waltham, MA, USA) with Al Ka radiation DIN physical characteristics in microspheres were analyzed by powder X-ray diffraction using an XRD spectrometer (Rigaku, Tokyo, Japan) Patterns were obtained between 5◦ and 90◦ (2θ/ min) at ambient temperature Materials and methods 2.1 Materials Dinotefuran technical concentrate (TC; 98%) was purchased from Shandong Lianhe Pesticide Co Ltd (Shandong, China); gelatin (water content ≤ 14%, pH 5–7) and chitosan (deacylation degree 80–95%, viscosity 50–800 mPa⋅s) were purchased from Sinopharm Chemical Reagent Co Ltd (Beijing, China); dinotefuran granules (commercial DIN; 1%) was purchased from Shandong Xinghe Co Ltd (Shandong, China) Other chemicals and reagents are commercially available and were used as received, without further purification Double-distilled Fig Schematic diagram of the spray-drying technology (A), and response surface plot showing the combined effects of X1 and X2 on Y (B) Q Zhang et al Carbohydrate Polymers 277 (2022) 118880 2.4 Heat storage stability maintain soil moisture at 20%, increasing to once daily watering when the number of leaves reached 12 The soil was collected 0.083, 1, 4, 7, 14, 21, 28, 35, and 42 days after application, and leaf samples were collected 14 days later Fruit samples were collected 77 and 84 days after application The samples were placed in appropriately labeled polythene bags and stored at − 20 ◦ C prior to analysis Detailed sample collection information is provided in the Supporting Information To evaluate storage stability, microspheres (2 g) were packed in glass tubes and stored at 54 ◦ C for 14 days (CIPAC, 2016; FAO, 2016) After removal from the tubes, the samples were allowed to return to room temperature (25 ± ◦ C) and changes in the microsphere surfaces were evaluated An analytical balance was used to weigh and calculate the mass loss High-performance liquid chromatography (HPLC) was used to measure the loading content of DIN 2.8 Control efficacy of DIN@CS-GEL against T vaporariorum 2.5 In vitro release behavior We used T vaporariorum (adults), which tends to occur naturally under greenhouse conditions, as a model organism to evaluate the in door control efficacy of DIN@CS-GEL Controls were as described in Section 2.7 The T vaporariorum (adult) population number was considered the population base 17 days after application Evaluations were performed 17, 21, 28, 35, and 42 days after application, giving a total of five investigations The number of live adults on the upper leaves and lower leaves were recorded The rate of insect population decline and the control efficacy were calculated using Eqs (3) and (4), respectively: Microspheres (0.1 g) were dispersed in a dialysis bag and immersed in 300-mL brown sample bottles containing the release medium The medium was maintained at different temperatures (10, 20, and 30 ◦ C) and pH values (5, 7, and 10) and magnetically stirred at 200 rpm At designated time intervals, mL of the solution was removed and replaced with the same volume of fresh solution to ensure a constant volume The DIN concentration in the solution was monitored using HPLC The microsphere release ratio was calculated using Eq (1) (Xiao et al., 2021): ∑t Mt Cumulative release percentage (%) = × 100 t=0 M Decrease rate of insects (%) = (1) Control efficacy (%) = 2.6 Leaching studies in soil Pt − P0 × 100 − P0 (4) where Pt and P0 are the decrease rates of T vaporariorum with and without treatment, respectively Red (Yunnan Province, P R China) and black soil (Heilongjiang Province, P R China) were air-dried, ground, screened (20-mesh), and packed into polyvinyl chloride columns (5 × 30, × 45, and × 60 cm2; 800, 1200, and 1600 g of soil) Pesticide (DIN TC, commercial DIN, and DIN@CS-GEL) was scattered onto the soil layer and overlain with 1-cmthick quartz sand Leaching was affected using 0.01 mol/L calcium chloride solution at 30 mL/h for 5, 10, 20, 30, and 40 h The soil columns were then cut into three even sections; the 30-cm columns were divided into sections of 0–10, 10–20, and 20–30 cm after leaching for 10 h and the 60 cm soil columns were divided into sections of 0–30, 30–45, and 45–60 cm after leaching for 40 h The loading content of DIN in each soil section and leaching solution were measured separately (USEPA, 2008) Each process was repeated thrice According to the DIN content in the soil and leaching solution of each section, the percentage of the total added amount was calculated using Eq (2): mi × 100 m0 (3) where N0 is the number of T vaporariorum in the uncontrolled plot, and Nt is the number in the treatment plot where Mt is the cumulative amount of DIN released at each sampling time point, t is the time of the release, and M0 is the initial weight of the DIN loaded in the sample Ri (%) = N0 − Nt × 100 N0 2.9 Loading content, encapsulation efficiency, and residue analysis 2.9.1 Loading content Microspheres (50 mg) were accurately weighed and extracted using methanol:water (25 mL; V/V = 20:80) via sonification (room tempera ture, 30 min; ultrasonic power 90 W) After centrifugation (8000 rpm, min) and filtration (0.45-μm filter membrane), the DIN concentration in the supernatant was evaluated by HPLC (1260-DAD, Agilent, Santa Clara, CA, USA) with an Agilent TC-C18 reversed-phase column (5 μm, 4.6 × 150 mm) and a diode array detector The mobile phase comprised methanol and water (20:80 v/v; mL/min flow rate) The analysis was performed at 270 nm, the maximum absorption wavelength of DIN The column temperature was maintained at 25 ◦ C (±0.5 ◦ C) and triplicate measurements were obtained Loading content and encapsulation effi ciency were calculated using Eqs (5) and (6) (Xu et al., 2021): (2) weight of DIN entrapped in microspheres × 100 weight of microspheres where Ri is the proportion of DIN content, mi is the DIN mass (mg) in each section of the soil and the leaching solution, and m0 is the total amount of DIN added (mg) Loading content (%) = 2.7 Determination of DIN residue in soil, leaves, and fruit Encapsulation efficiency (%) = We prepared DIN@CS-GEL/soil mixtures to study the DIN trans location, distribution, and degradation rates in cucumber plants and soil DIN@CS-GEL and commercial DIN contained 150 g active in gredients/ha (soil and pesticide mixed thoroughly, following which seeds were sowed); the commercial DIN granules were used as control agents and an untreated soil constituted the blank control group (CK) In the early planting stage, the cucumbers were watered for 2–3 days to (5) weight of DIN entrapped in microspheres ×100 initial weight of DIN employed (6) 2.9.2 Residue analysis Extraction and purification procedures were based on the simple and effective QuEChERS method (Lombardo-Agüí et al., 2015) Sample pretreatment mainly incorporated acetonitrile extraction, PSA, C18, anhydrous MgSO4 purification, and Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis Q Zhang et al Carbohydrate Polymers 277 (2022) 118880 (A1) (A2) (D) (G1) (G2) (B) (C) (E) (F) (H) (I) Fig SEM images of (A1) DIN@GEL (GEL:CS 1:0), (B) local surface amplification of DIN@GEL, and (C) inner structure of DIN@GEL Different ratios of GEL and CS: (D) 7:1, (E) 5:1, and (F) 3:1 SEM images of (G1) DIN@CS-GEL (GEL:CS 1:1), (H) local surface amplification of DIN@CS-GEL, (I) inner structure of DIN@CS-GEL Inserts: size and distribution of DIN@GEL (A2) and DIN@CS-GEL (G2) Detailed pretreatment processes and instrumental analysis conditions are provided in the Supporting Information stability of the microspheres was obtained at a GEL:CS ratio of 1:1, resulting in the achievement of a uniform powder without lumps (Fig 3E) Thus, the introduction of CS as composite carriers improved the thermal stability of DIN@GEL (Table S4), potentially owing to the high melting point of CS Consequently, DIN@CS-GEL (1:1) was used for subsequent experiments The composition of polymeric dispersions and the spray-drying conditions (e.g., pump rate, inlet temperature, and air pressure) strongly affect the relevant characteristics of microspheres (Zhang et al., 2019) To optimize the spray-drying parameters, we evaluated the in fluence of the pump rate (X1) and inlet temperature (X2) on the loading content (Y) (Table S5) The Y response variables were best fitted by the quadratic model The equation depicting the relation between the in dependent and response variables derived using multiple regression analysis is expressed as follows: 2.10 Statistical analysis Statistical analyses were performed using the SPSS 22 software (IBM, Armonk, NY, USA), and data are presented as the mean ± standard deviation Differences between treatments were analyzed using the Duncan's multirange test and independent sample t-test, and a p value < 0.05 indicated statistical significance Results and discussion 3.1 Preparation of DN@CS-GEL Dinotefuran-loaded gelatin microspheres (DIN@GEL) exhibited a Y = − 28.58250 + 1.38625X1 + 0.536375X2 − 0.004875X1 X2 − 0.10625X1 − 0.001458X2 collapsed hollow structure under SEM (Fig 2A1 and B) and demon strated substantial agglomeration following heat storage (54 ◦ C) for 14 days (Fig 3A), likely owing to the low melting point of GEL Therefore, we introduced CS to improve the microsphere heat storage performance Fig depicts the appearances of microspheres with different GEL:CS ratios (1:0, 7:1, 5:1, 3:1, and 1:1) after heat storage The optimal storage Summary statistics of the analysis of variance (ANOVA) results (Table S6) indicate that Y was significantly influenced by X1, X2, X12, and X22 The models were further analyzed to evaluate the significance of the response surface models (Fig 1B) X1 exerted a more significant effect than X2 on Y, which can be attributed to X1 directly affecting the Q Zhang et al Carbohydrate Polymers 277 (2022) 118880 (B) (A) (C) (D) (E) Fig Different ratios of GEL and CS (A) 1:0, (B) 7:1, (C) 5:1, (D) 3:1, and (E) 1:1 after storage (54 ◦ C, 14 days) atomization effect during spray-drying When the gear of pump rate was 1, there was a high loading content but the output was extremely low Hence, the optimized spray-drying parameters were as follows: inlet temperature 170 ◦ C, outlet temperature 75 ◦ C, and the gear of pump rate was The loading content and encapsulation efficiency of DIN@CS-GEL were 21.5% and 98.17%, respectively microspheres were rough and wrinkled (Figs 2D–F and S2) because of the rapid water evaporation at high temperatures during spray-drying The modification effect of CS on DIN@GEL smoothed the microsphere surface (Fig 2H) and changed the microsphere structure from hollow to solid (Fig 2C and I) Smoothness increased with an increasing CS con centration and particle size decreased with an increasing CS concen tration (Fig S2) A plausible mechanism for the changes in morphology is as follows: the GEL molecular chain has excellent flexibility and readily shrinks; heating changes the GEL tertiary structure, causing the triple helix GEL molecules to unfold and exposing the internal amino acid residue (Gong et al., 2008) CS physically modifies GEL, with hydrogen bond formation between CS–NH2 and GEL–COOH (Cheng et al., 2010; Cui et al., 2015) The interaction points of GEL and CS 3.2 Microsphere characterization 3.2.1 SEM of microspheres Different surface morphologies were observed for microspheres with different GEL:CS ratios via SEM, with the microspheres being nearly monodispersed (Fig 2A1 and G1) At low CS concentrations, the Fig FT-IR spectra (A), and XPS wide scans (B) of different samples: high-resolution carbon spectra (C), oxygen spectra (D), and nitrogen spectra (E) of DIN@CSGEL; XRD spectra (F), DTG (G), TGA (H), and DSC (I) curves of different samples Q Zhang et al Carbohydrate Polymers 277 (2022) 118880 molecular chains increased so that the force also gradually increased The chitosan and gelatin molecules further entangle to fill gaps and gradually change from hollow to solid microspheres, rendering the structure more compact These mechanisms could also potentially explain the smaller particle size of DIN@CS-GEL than DIN@GEL The CS molecular chain has a cyclic structure with strong rigidity (Zhang & Yao, 2006), which likely limits the surface collapse of gelatin microspheres to form a smooth and solid sphere This is consistent with the literature, which reported an enhancement of membrane mechanical properties using a combination of CS and GEL (Gong et al., 2008) Notably, neither the DIN nor GEL/CS crystals were visible in the microsphere images, indicating complete incorporation of DIN inside the microsphere mesh structure The diameter of DIN@CS-GEL (1:1) was 9.117 μm (D50 = 8.571; D90 = 14.95, SPAN = 1.270) (Fig 2G2); that of DIN@GEL was 10.77 μm (D50 = 8.464; D90 = 20.00, SPAN = 1.968) (Fig 2A2) In summary, we observed a relatively uniform particle size distribution of DIN@CS-GEL (Demina et al., 2012) The O1s spectrum showed two binding energy – O/C–O–C) and 532.28 eV (C–O) (Huang et al., peaks at 530.98 (C– 2012), and the N1s spectrum showed three binding energy peaks at – C–N), and 401.39 eV (C–NH3+/ 399.38 (–NH2/–NH–), 400.38 (O– C–N–C) in Fig 4D–E These results are consistent with the previous characterization The physical nature of the microspheres was further confirmed by XRD Sharp characteristic peaks resulting from the crystalline nature of DIN were much less intense or absent in DIN@CS-GEL (Fig 4F), indi cating that the microsphere-entrapped DIN was dispersed and amor phous (Saravanan et al., 2011) 3.2.3 Thermal properties of DIN@CS-GEL Consistent with the results of DTG (Fig 4G), TGA analysis showed that the mass change of DIN occurred in the range of 500 ◦ C, with the mass loss rate rising rapidly at 210 ◦ C (Fig 4H) The process was divided into three stages, i.e., 25–210, 210–340, and 340–500 ◦ C Weight loss below 210 ◦ C was caused by water evaporation and GEL decomposition; the mid-stage was related to DIN and partial CS decomposition, and the third processes mainly corresponded to the decomposition of the remaining carriers (Lin et al., 2013) This suggested that DIN was embedded into the carriers and did not react chemically therewith because no new characteristic thermal decomposition peak emerged over the DIN@CS-GEL thermal decomposition process beyond those of CS–GEL and DIN In the DSC results, the characteristic DIN melting peak (107.84 ◦ C) did not appear in DIN@CS-GEL, indicating that DIN was dispersed in the microspheres in an amorphous rather than crystalline form (Fig 4I), consistent with the XRD results The presence of DIN did not substantively alter the microsphere melting temperature, indicating that DIN was no chemical reaction wrapped in the carrier 3.2.2 FT-IR, XRD, and XPS analyses FT-IR measurements were performed to investigate component in teractions and the presence of functional groups in the system GEL exhibited characteristic peaks at 1431.89 cm− 1, attributed to symmetric –COOH group stretching (Fig 4A) Moreover, the intense peak at – O stretching vibrations (Wang et al., 1628.11 cm− was assigned to C– 2013) For CS, peaks at 1073.18 cm− were attributed to the C–O–C antisymmetric stretching (Subramanian et al., 2014) CS-GEL displayed characteristic peaks of both CS (1072.23 cm− 1) and GEL (1630.52 and 1425.85 cm− 1), indicating that the two were well-mixed (Peng et al., 2020) Compared with blank microspheres, the characteristic DIN peak at 1315.72 (–NO2) was observed in DIN@CS-GEL, confirming the successful loading of DIN into the microspheres As the microsphere GEL content increased, the –NH2 absorption peak at 1550.55 cm− gradu ally weakened owing to the strong CS-GEL hydrogen bonding (Fig S3) CS showed good compatibility with GEL in the microsphere system and physically modified DIN@GEL successfully (Dong et al., 2004; Wang et al., 2018) The bonding of CS and GEL was simulated via dynamic relaxation using Materials Studio Forcite (Fig 5) XPS provided information concerning the chemical elements on the microsphere surface, revealing the related functional groups obtained by fitting C1S, O1S, and N1S peaks When modified with CS-GEL, the 285.48 (C1S), 531.45 eV (O1S), and 399.86 (N1S), peaks of DIN@CS-GEL were more intense than those of DIN, owing to the introduction of ele ments C, N, and O, which indicated that DIN was coated with CS-GEL on the surface (Wang et al., 2020; Xu et al., 2018) in Fig 4B As indicated in Fig 4C, high-resolution carbon spectra exhibited three types of carbon – O) bonds: 284.59 (C–C/C–H), 285.58 (C–O), and 287.48 eV (C– 3.3 Controlled release of DIN in vitro 3.3.1 Temperature-responsive release Temperature-controlled release profiles were exploited to assess the thermal stimulus responsiveness of the DIN-loaded microspheres Initial burst releases reached 84.99 ± 5.77% at 30 ◦ C in the first 15 h, whereas cumulative release reached only 47.43 ± 1.81% and 65.85 ± 2.39% at 10 and 20 ◦ C, respectively, after 29 h (Fig 6A) Temperatures above 20 ◦ C promoted GEL molecular chain extension and extensive void formation in the microspheres and thus the swelling properties, with reduced intermolecular hydrogen-bonding interactions (Cheng et al., 2010) and increased GEL solubility The thermal motion of DIN mono mers consequently accelerated, promoting DIN diffusion and migration outside the microspheres into the medium Conversely, the intra- and intermolecular CS-GEL interactions below 20 ◦ C promoted the formation Fig Plausible interactions between GEL and CS Q Zhang et al Carbohydrate Polymers 277 (2022) 118880 Fig Release profiles of DIN@CS-GEL (A) fitted plots using the Peppas model (B) at different temperatures Release profiles of DIN@CS-GEL (C) fitted plots using the Higuchi model (D) at different pH values Schematic diagram of DIN@CS-GEL release behavior at various temperature and pH values (E) of hydrogen bonds within and between molecules (Cheng et al., 2010) Therefore, DIN release could be efficiently adjusted by temperature owing to the temperature-responsive DIN@CS-GEL structure The schematic diagram of release behavior is shown in Fig 6E DIN release from the microspheres was most consistent with the Peppas equation (Table S7); Fig 6B illustrates the fitted plots Regarding the Peppas model, n indicates the release mechanism; where the n value of less than 0.43 at 30 ◦ C and 20 ◦ C indicates that the DIN release from the microspheres follows the Fickian diffusion (Ritger & Peppas, 1987) In other temperature-controlled pesticide release systems, it is reported that the cumulative active ingredient release from fibrous GEL hydrogels was the highest at 40 ◦ C (87%), followed by 37 and 25 ◦ C (Zhang et al., 2021) Alternatively, a previous study on CS-GEL-glycerol phosphate hydrogels reported that the thermosensitive can be adjusted by adding gelatin (Cheng et al., 2010) This provides a theoretical basis for adjusting the carrier release rate according to external environmental factors 3.3.2 pH-responsive release Soil is weakly acidic, neutral, or basic Previous simulation studies employed pH values of 5, 7, and to evaluate K2SO4 release (Chen & Chen, 2019) and pH values of 4.0, 7.0, and 9.2 to investigate Q Zhang et al Carbohydrate Polymers 277 (2022) 118880 thiamethoxam release from boric acid-crosslinked carboxymethyl cel lulose hydrogel-based formulations (Sarkar & Singh, 2019) Thus, release media with pH values of 5, 7, and 10 were selected to investigate DIN@CS-GEL controlled-release profiles The schematic diagram of release behavior is shown in Fig 6E DIN exhibited sustained release from microspheres, with cumulative release reaching 70.72–95.80% after 11 h (Fig 6C) and 61.54 ± 1.43% and 69.01 ± 2.18% at pH and 10 after h, respectively but only 47.33 ± 1.76% at pH This indicates that DIN@CS-GEL released more slowly in acidic media than in basic and neutral media, likely because the protons (H+) decrease the degree of –COOH dissociation in acidic solutions, leading to the shrinkage of DIN@CS-GEL polymer chains and the closure of microsphere pores (Xu et al., 2018) Under alkaline conditions, the GEL structure was loose and relatively more free amino groups were observed on the CS molecules, thereby weakening the CS-GEL interactions, enhancing the ionization of the –COOH group, and leading to maximum microsphere swelling (Xu et al., 2021) and rapid DIN release Moreover, the swelling behavior of DIN@CS-GEL (Fig S4) increased with GEL content (Mir et al., 2019) The correlation coefficient R2 was established for evaluating the release mechanism The data best fitted the Higuchi model (Table S8), achieving the maximum R2 values; the equation was Q = at1/2 + b (Fig 6D) Some carriers can readily produce different release effects by altering the external pH medium According to a previous report, a high pH response was observed for GEL-polyvinyl alcohol composite hydrogels at pH 1.2 (Akhlaq et al., 2021) In contrast, Xu et al prepared carbox ymethyl CS hydrogels that showed high pH responses at pH greater than 7.5 (Xu et al., 2021) Similarly, the carrier release performance char acteristics of the CS-GEL system developed in this study can be exploited to adjust the release rate according to different external environmental factors, highlighting its potential for future development as an efficient controlled release system 31270.5-2014 guidelines for the classification of the mobility of pesti cides in soil (Table S9), the leaching solution (R4) of DIN TC and com mercial DIN accounted for more than 50% of the DIN in 0–30 cm soil columns after 10 h, indicating marked leaching propensity; however, the DIN@CS-GEL leaching solution and the 20–30 cm soil layer (R4 + R3) accounted for more than 50% of DIN, revealing moderate leaching In the 30, 45, and 60 cm soil columns, 37.61–98.36%, 27.99–80.00%, and 22.36–67.15% of DIN TC active ingredients and 30.91–79.14%, 17.29–50.41%, and 9.36–32.09% of commercial DIN active ingredients migrated into the leachate, respectively (Fig 7A–C) Conversely, most DIN remained in the column layers for DIN@CS-GEL (Fig 7D–E), reflecting 13.61–46.36% and 5.57–19.89% reduced leaching compared with DIN TC and commercial DIN, respectively Black soil exhibited 1.35–16.04% less leaching than red soil Introducing carriers is a feasible method to reduce pesticide leaching in soil For example, decreased atrazine mobility in soil was achieved using carboxymethyl CS-bentonite controlled release formulations (Hu et al., 2012), and a 3D network-structured hydrogel with gentian violet incorporated into biochar achieved controlled release along with decreased leaching loss (Xiang et al., 2020) Zhang and Yao (2006) re ported that the CS molecule is rigid and has good supporting properties, while GEL is a flexible molecule with swelling properties; the combi nation of GEL and CS improves the mechanical properties of the film, which implies that composite carriers probably modify the structures of microspheres Microspheres swell with water in the soil and have a certain mechanical support strength to fill the pores of the soil which DIN@CS-GEL is immobilized in the soil Thus, our strategy to alter the pesticide formulation by introducing CS-GEL to reduce the leaching loss of hydrophilic DIN is expected to be useful for reducing reduce the risk of DIN groundwater pollution and may constitute an approach for pro tecting the groundwater resources 3.4 Retarded leaching of DIN in the soil column 3.5 Residue analysis The leaching performance of DIN@CS-GEL, DIN TC, and commercial DIN was evaluated in a simulated soil column According to the GB/T 3.5.1 Degradation dynamics of DIN residues in soil The residual amount of DIN in soil showed an exponential Fig Samples leaching in simulated leaching columns of 30 (A), 45 (B), and 60 cm (C) after 40 h Sample distribution in soil in simulated leaching columns after 10 h (D) and 40 h (E) Q Zhang et al Carbohydrate Polymers 277 (2022) 118880 Fig Degradation rate of DIN@CS-GEL and commercial DIN in soil on different days (A) and accumulation of DIN in upper and lower leaves, corresponding to the concentration of DIN in the soil on different days (B) Control efficacy of DIN on T vaporariorum in upper and lower leaves (C) Schematic diagram of release and insecticidal activity of DIN@CS-GEL and commercial DIN (D) Independent sample t-tests were used to analyze the differences in the concentration of DIN in soil and control efficacy of T vaporariorum between DIN@CS-GEL and commercial DIN Duncan's multirange test was used to analyze the DIN in the upper and lower leaves, corresponding to DIN@CS-GEL and commercial DIN Different letters indicate significant differences between values (P < 0.05) leaves, reaching the maximum value after 35 days With the same application dose, the DIN accumulation in leaves from DIN@CS-GEL was 7.56–26.09% higher than that from commercial DIN During 14–42 days, the degradation rate of commercial DIN in soil exceeded that of the same application dose of DIN@CS-GEL by 15.31–35.18%, whereas DIN accumulation in leaves was slightly lower than that of DIN@CS-GEL, likely owing to the shorter half-life (14 days) of com mercial DIN compared with that of DIN@CS-GEL in soil, being subject to ready degradation and metabolic use by the soil microecological envi ronment At 42 days, DIN accumulation in leaves from DIN@CS-GEL remained 19.42–22.81% higher than that from commercial DIN, which ensured the efficacy of cucumber against T vaporariorum in the later growth period relationship with the time interval after application (t) and the degra dation dynamics followed the first-order kinetic equation Ct = C0e− KT (Table S10) Under the same application dose, the degradation rate of DIN@CS-GEL in the soil was slower than that of the commercial DIN (by 10.87–35.18%) in Fig 8A and the degradation half-life was prolonged by 11 days Therefore, introducing CS-GEL not only affords the controlled release of DIN but also decreases its degradation rate in soil, improving DIN usage 3.5.2 DIN accumulation in cucumber leaves and fruits With increasing application time, DIN accumulation first increased and then decreased in cucumber leaves (Fig 8B) The DIN accumulation in the lower leaves was 12.57–28.63% higher than that in the upper (A1) (A2) (A3) (B1) (B2) (B3) (C1) (C2) (C3) (D1) (D2) (D3) Fig Representative images illustrating control efficacy against T vaporariorum on the upper (A) and lower (B) leaves after 17 days, and on the upper (C) and lower (D) leaves after 35 days (1: blank control; 2: commercial DIN; 3: DIN@CS-GEL) Q Zhang et al Carbohydrate Polymers 277 (2022) 118880 To evaluate the long-term performance of DIN@CS-GEL, we collected cucumber fruit samples for residue analysis on days 77 and 84 (harvest period) after application The maximum residue limit values of DIN in cucumbers were set at mg/kg by GB 2763–2016 and 0.5 mg/kg by EFSA (EFSA, 2013) Following this, the residue in the cucumber fruits was below the limit of quantification (0.01 mg/kg), and thus far below the maximum residue limit, indicating a very low risk of residual DIN in the cucumber fruit during the harvest period and confirming the safety of DIN@CS-GEL Conceptualization, Supervision Manli Yu: Conceptualization, Re sources Lirui Ren: Formal analysis, Software, Methodology Yongfei Guo: Supervision, Conceptualization Qinghua Li: Supervision, Conceptualization Mingming Yin: Project administration, Visualiza tion Xiaolong Li: Conceptualization Fuliang Chen: Funding acquisi tion, Supervision, Visualization Declaration of competing interest The authors declare no competing financial interest 3.6 Control efficacy of DIN@CS-GEL against T vaporariorum Acknowledgments T vaporariorum is one of the most common insect pests in green houses (Fattoruso et al., 2021) Compared with commercial DIN, DIN@CS-GEL showed favorable control efficacy against T vaporariorum (Fig 9) The schematic diagram of release behavior is shown in Fig 8D The control efficacy exhibited 1.78–6.86% higher efficacy against T vaporariorum in the lower leaves of cucumber than in the upper leaves, with both reaching their maximum values after 35 days (Fig 8C), consistent with the results in Section 3.5.2 The control efficacy of DIN@CS-GEL at 21–42 days differed significantly from that of com mercial DIN at the same dose, being 16.85–26.79% higher and average control efficacies of 83.54% and 64.81%, respectively, at 42 days (Table S11) This favorable control efficacy likely results from the pro tective and immobilization effects of composite carriers toward DIN, leading to reduced soil leaching and an improved pesticide utilization rate Recently, Guo et al (2021) reported that, for non-sustained release formulations of clothianidin, active ingredients were entirely exposed to the environment and were rapidly released, whereas composite carriers efficiently contained clothianidin, yielding control efficacy against pests and facilitating persistence Together, these results indicate that the modification of pesticide formulations by introducing composite carriers to enhance efficacy offers considerable potential to reduce pesticide use for sustainable plant protection This work was supported by a grant from the National Key Research and Development Program of China (grant number 2016YFD0200500) Appendix A Supplementary data Supplementary data to this article can be found online at https://doi org/10.1016/j.carbpol.2021.118880 References Akhlaq, M., Azad, A., Ullah, I., Nawaz, A., Safdar, M., Bhattacharya, T., et 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as a drug carrier for the delivery of bis-desmethoxy curcumin analog Carbohydrate Polymers, 114, 170–178 11 ... agricultural application of microsphere delivery systems (Grillo et al., 2011; Zhang et al., 2019), to the best of our knowledge, microspheres based on biodegradable CS-GEL to reduce dinotefuran leaching. .. eV (C– 3.3 Controlled release of DIN in vitro 3.3.1 Temperature-responsive release Temperature -controlled release profiles were exploited to assess the thermal stimulus responsiveness of the DIN-loaded... (Sarkar & Singh, 2019) Thus, release media with pH values of 5, 7, and 10 were selected to investigate DIN@CS-GEL controlled- release profiles The schematic diagram of release behavior is shown in