Controlled release of dinotefuran with temperature/pH-responsive chitosan-gelatin microspheres to reduce leaching risk during application

Carbohydrate Polymers 277 (2022) 118880

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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.

1. 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: (F. Chen).
/>Received 25 June 2021; Received in revised form 4 November 2021; Accepted 8 November 2021
Available online 13 November 2021
0144-8617/© 2021 The Authors.
Published by Elsevier Ltd.
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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 ±
1 ◦ 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 8 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 2 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.

2. 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. 1. Schematic diagram of the spray-drying technology (A), and response surface plot showing the combined effects of X1 and X2 on Y (B).
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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 ± 1 ◦ 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, 1 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
0

Decrease rate of insects (%) =

(1)

Control efficacy (%) =

2.6. Leaching studies in soil

Pt − P0
× 100
1 − 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, 5 × 45, and 5 × 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, 5
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; 1 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.

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Carbohydrate Polymers 277 (2022) 118880

(A1)

(A2)

(D)

(G1)

(G2)

(B)

(C)

(E)

(F)

(H)

(I)

Fig. 2. 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.
3. 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 2 − 0.001458X2 2

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. 3 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
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Carbohydrate Polymers 277 (2022) 118880

(B)

(A)

(C)

(D)

(E)

Fig. 3. 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 5. 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. 4. 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.
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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− 1 was assigned to C–
2013). For CS, peaks at 1073.18 cm− 1 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− 1 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. 5. Plausible interactions between GEL and CS.

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Carbohydrate Polymers 277 (2022) 118880

Fig. 6. 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 9 to evaluate K2SO4 release (Chen &
Chen, 2019) and pH values of 4.0, 7.0, and 9.2 to investigate
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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 7 and
10 after 5 h, respectively but only 47.33 ± 1.76% at pH 5. 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. 7. 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).
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Carbohydrate Polymers 277 (2022) 118880

Fig. 8. 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. 9. 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).
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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 2 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.
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4. Conclusions
We successfully fabricated GEL microspheres physically modified
with CS to develop a pH- and temperature-responsive controlled-release
system for the hydrophilic pesticide DIN, which was encapsulated into
microspheres via spray-drying technology. DIN@CS-GEL was charac­

terized using integrated methods to elucidate its formation mechanisms.
Optimized spray-drying parameters were determined using 3D surface
plots as follows: inlet temperature 170 ◦ C; outlet temperature 75 ◦ C, and
the gear of pump rate was 5. The loading content was 21.5%, the
encapsulation efficiency rate was 98.17%, and the particle size was 9.12
μm. In vitro release experiments revealed that DIN@CS-GEL enabled
marked pH and temperature responsiveness, a feature suitable for the
intelligent controlled release of DIN. Moreover, 5.57–19.89% and
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compared with commercial DIN and DIN TC, respectively. The biode­
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DIN in soil, thereby significantly reducing pesticide leaching. Moreover,
DIN@CS-GEL lengthened the half-life of DIN in soil and increased its
cumulative absorption in leaves. The ultimate fruit residue was far
below the maximum residue limits while enabling enhanced efficacy
against T. vaporariorum. These results demonstrate that modification of
the pesticide formulation constitutes a promising, simple, and low-cost
strategy with the potential to develop approaches for controlled pesti­
cide release, thereby decreasing pesticide leaching loss and improving
pesticide usage efficiency.
CRediT authorship contribution statement
Qizhen Zhang: Experiment, Conceptualization, Data curation,
Methodology, Validation, Investigation, Writing - original draft. Yu Du:
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