Stability and Biological Activity Evaluation of Chlorantraniliprole Solid Nanodispersions Prepared by High Pressure Homogenization
Stability and Biological Activity Evaluation of Chlorantraniliprole Solid Nanodispersions Prepared by High Pressure Homogenization
Bo Cui 0 1
Lei Feng 0 1
Chunxin Wang 0 1
Dongsheng Yang 0 1
Manli Yu 0 1
Zhanghua Zeng 0 1
Yan Wang 0 1
Changjiao Sun 0 1
Xiang Zhao 0 1
Haixin Cui 0 1
0 Institute of Environment and Sustainable Development in Agriculture, Chinese Academy of Agricultural Sciences , Beijing , China
1 Editor: Etsuro Ito, Waseda University , JAPAN
Poorly water-soluble compounds are difficult to develop as pesticide products and face great challenges in water-based and environmentally friendly formulation development. In this study, high pressure homogenization combined with lyophilization was adopted to prepare the solid nanodispersions of chlorantraniliprole with poor solubility and high melting point. The mean particle sizes of the solid nanodispersions with different pesticide contents were all less than 75 nm, even when the content was up to 91.5%. For the 2.5% chlorantraniliprole solid nanodispersion with the mean particle size of 29 nm, the suspensibility and wetting time in water were 97.32% and 13 s, respectively. The re-dispersibility and wettability were superior to those of conventional water dispersible granules. The retention on the rice leaf of 18.7 mg/cm2 was 1.5 and 3 times that of commercial aqueous suspension concentrate and pure water. The bioassay result to diamondback moths indicated that the toxicity of the solid nanodispersion was 3.3 and 2.8 times that of technical and aqueous suspension concentrate, respectively. Moreover, the solid nanodispersion has the advantages of total avoidance of organic solvents, significant reduction of surfactants and feasibility of obtaining high concentration nanoformulations. The solid nanodispersion is an attractive candidate for improving pesticide solubility and efficacy, and its application in crop production will reduce both residues in food and environmental pollution of pesticide.
Data Availability Statement: All relevant data are
within the paper.
Funding: This research was supported by Major
National Scientific Research Program of China (No.
2014CB932200) and Basic Scientific Research
Foundation of National non-Profit Scientific Institute of
China (No. BSRF201406). The funders had no role in
study design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing Interests: The authors have declared
that no competing interests exist.
Pesticide as an important kind of agrochemicals has been widely applied in the world for plant
protection and decreasing production loss [
]. However, most of effective pesticide compounds
have poor solubility in water which limits the development of their formulations with high
efficacy and safety as stated in our previous article [
]. The conventional pesticide formulations
like wettable powder (WP) and emulsifiable concentrate (EC) have disadvantages of dust drift,
overuse of organic solvent and low efficacy, which lead to expensive cost and environmental
]. Recently, nanotechnology has been exploited to solve the above problems.
According to the Ostwald-Freundlich and Noyes-Whitney equations, the saturation solubility
and dissolution rate increase with decreasing particle size [
]. In addition, the larger surface
area of nanoparticles can also improve the coverage, adhesion and penetration of particles to
the surface of crop leaves and targeted organisms, and further enhance the bioavailability of
pesticide as described in other literatures [
The main techniques for producing nanoformulations involve the top-down method such
as wet-milling and high pressure homogenization (HPH) [
], and the bottom-up method
like microprecipitation and supercritical fluid [
]. Compared to the bottom-up
technology, almost any compound with poor solubility can be processed with the top-down process,
despite of being poorly soluble in aqueous and simultaneously in non-aqueous media . The
HPH process refers to passing a coarse dispersion into a vessel through a very small
homogenization gap. High velocity and pressure induce shear force, cavitation force and particle collision
to break big particles into small ones [
]. The advantages of HPH technology are free of
organic solvent, ease of scale up and avoidance of contamination with erosion from milling
pearls during the wet-milling process. This technology has been extensively used in the food,
pharmaceutical and biotechnology industries . Nevertheless, relevant reports in the field of
agriculture are rare.
The nanoformulation of pesticide has attracted extensive attention and the corresponding
formulations including microemulsion, nanoemulsion and nanosuspension have been
]. However, the above formulations are all liquid form and have problems of
stability for the nanosuspension and heavy use of surfactant for the self-emulsifying systems. A
solid nanodispersion is a nanoformulation with hydrophobic pesticide compound dispersed in
a solid hydrophilic matrix as described in detail previously . It can not only maintain the
desirable solubility and dispersibility of water-based nanoformulations, but also decrease
surfactant content and improve the stability and safety during storage and transportation.
Chlorantraniliprole, [3-bromo-N-[4-chloro-2-methyl-6-[(methyl amino) carbonyl]
phenyl]-1-(3-chloro-2-pyridinyl)-1H-pyrazole-5-carboxamide], is a new anthranilic diamide
pesticide developed by DuPont Crop Protection (Fig 1) [
]. As described previously, it has a
novel mode of action called ryanodine receptor activators which can lead to the depletion of
intracellular calcium stores and cause impaired muscle regulation, paralysis and insect death
]. The pesticide exhibits remarkable insecticidal efficacy and mammalian safety, and has
been widely used for pest control in agriculture [
]. The current dominant formulations
for chlorantraniliprole are aqueous suspension concentrate (SC) and water dispersible granule
(WG). The construction of the chlorantraniliprole solid nanodispersion may further improve
the pesticide stability, bioavailability and transportation convenience.
In our previous work, a solid nanodispersion of lambda-cyhalothrin was developed based
on melt-emulsification and high-speed shearing methods which were suitable for pesticide
compounds with melting points lower than 100°C [
]. In this present investigation, high
pressure homogenization combined with lyophilization was successfully applied to prepare the
solid nanodispersions of chlorantraniliprole with poor solubility and high melting point (208–
210°C). This method is applicable for poorly water-soluble pesticides regardless of their
melting points. The chlorantraniliprole solid nanodispersion was characterized with respect to
crystallinity, suspensibility, wettability, retention, stability and biological activity. This solvent-free
nanoformulation substantially reduced the surfactant content relative to conventional
formulations and enhanced the safety and environmental friendliness of pesticide. In addition, the
high concentration nanoformulation with 91.5% pesticide could be obtained using this method
and has important application prospect for ultra-low volume and aerial spray.
2 / 16
Fig 1. Chemical structure of chlorantraniliprole.
2. Materials and Methods
Chlorantraniliprole (96%) and standard hard water (ρ(Ca2+ + Mg2+) = 342 mg/l) were
obtained from China Agricultural University. 1-Dodecanesulfonic acid sodium salt (SDS, 99%,
CAS number: 2386-53-0), sodium ligninsulfonate (SL, CAS number: 8061-51-6),
polyvinylpyrrolidone K90 (PVP K90, 90%, CAS number: 9003-39-8, average molecular weight: 360000),
polyethylene glycol mono-4-nonylphenyl ether (PGME, n 15, CAS number: 26027-38-3),
hexadecyltrimethylammonium bromide (CTAB, 98%, CAS number: 57-09-0) and sucrose
(99%, CAS number: 57-50-1) were purchased from J&K Scientific Ltd. (Beijing, China).
Hydroxypropyl methylcellulose (HPMC, CAS number: 9004-65-3, viscosity: 2600–5600 cP) was
obtained from Sigma-Aldrich Shanghai Trading Co., Ltd. (Shanghai, China). Maleic
rosinpolyoxypropylene-polyoxyethylene ether sulfonate (MRES, SP-SC29, number-average
molecular weight: 1300) and polycarboxylate (SP-2728, number-average molecular weight: 13200)
were provided by Sinvochem S&D Co., Ltd. (Jiangsu, China). The chlorantraniliprole SC (200
g/l, CORAGEN) and WGs (35%, ALTACOR and 35%, JIATENG) were purchased from
DuPont Agricultural Chemicals Ltd. (Shanghai, China). The chlorantraniliprole granule (GR,
0.4%, FERTERRA) was bought from Sinon Chemical (China) Co., Ltd. (Shanghai, China). All
the chemicals were used as received.
2.2. Preparation of the Chlorantraniliprole Solid Nanodispersions
The preparation processes of the solid nanodispersions with 2.5%, 7.5%, 22.5% and 67.5%
chlorantraniliprole were almost exactly the same except for the adding amount of sucrose.
3 / 16
Taken the 2.5% chlorantraniliprole solid nanodispersion as an example, the detailed
preparation procedure was as follows. Firstly, 0.076 g MRES and 0.076 g polycarboxylate were
dissolved in 58 ml water to get a colorless and transparent solution by stirring at 800 rpm for 5
minutes on a magnetic stirrer (RCT Basic, IKA1-Works Guangzhou, Guangzhou, China).
After adding 3.166 g chlorantraniliprole into the above solution, the mixture was further stirred
at 800 rpm for 5 minutes by a magnetic stirrer (RCT Basic, IKA1-Works Guangzhou,
Guangzhou, China) and emulsified at 10000 rpm for 15 minutes by a shearing machine (C25, ATS
Engineering Ltd., Vancouver, Canada) to make the pesticide particles uniformly suspend in the
suspension. Secondly, the prepared suspension was subjected to a high pressure homogenizer
(AH-100D, ATS Engineering Ltd., Vancouver, Canada) and homogenized at 300 bar, 600 bar,
900 bar, 1200 bar and 900 bar for 10 cycles at each pressure to obtain a chlorantraniliprole
nanosuspension. Here, a cycle meant that the entire aqueous dispersion passed through the
homogenizer once. This definition was the same as that in other literatures [
stirring at 800 rpm on a magnetic stirrer (RCT Basic, IKA1-Works Guangzhou, Guangzhou,
China), 118.242 g sucrose was added slowly. Then water was removed using a freeze drier
(FD81, EYELA, Tokyo, Japan) to obtain the 2.5% chlorantraniliprole solid nanodispersion.
The difference between the preparation of the 91.5% chlorantraniliprole solid
nanodispersion and the above procedure was that sucrose was not added in the process. Firstly, the
nanosuspension with 0.076 g MRES, 0.076 g polycarboxylate and 3.166 g chlorantraniliprole was
produced according to the above method. Then the aqueous nanosuspension was directly
lyophilized by a freeze drier (FD-81, EYELA, Tokyo, Japan) to acquire the solid nanodispersion
with 91.5% pesticide.
2.3. Particle Size and Zeta Potential Measurements
The particle size, polydispersity index (PDI) and zeta potential of the samples were
characterized at 25°C using a Zetasizer Nano ZS 90 (Malvern, Worcestershire, UK). Particle size
measured by dynamic light scattering (DLS) was expressed by the mean size and 90% diameter
percentile (D90). Each sample was measured in triplicate. All sizes and PDIs were recorded as
mean ± standard deviation (S.D.).
2.4. Morphological and Structural Characterizations of the Nanoparticles
The morphological characterizations of the nanoparticles were performed using a scanning
electron microscope (SEM) and a transmission electron microscope (TEM). SEM imaging was
conducted by a scanning electron microscope (JSM-7401F, JEOL, Tokyo, Japan) at 3 kV. 3 μl
of the aqueous dispersion with re-dispersed nanoparticles was dropped on a freshly cleaned
silicon slice. The sample was air-dried and coated with platinum (thickness 2 nm) by a sputter
coater (Beijing Elaborate Technology Development Ltd., Beijing, China) using an electric
current of 2 mA for 3 minutes. The images were recorded at low electron image (LEI) mode and
the work distance (WD) was 8.1 mm.
The morphology of the nanoparticles was also visualized by a transmission electron
microscope (HT7700, HITACHI, Tokyo, Japan). 3 μl of the sample dispersion was dropped on a
copper grid. The grid was left overnight for complete dryness before TEM imaging at 80 kV and
X-ray diffraction (XRD) was applied to evaluate the crystallinity of the samples by a
diffractometer (D8 ADVANCE, Bruker AXS Inc., Karlsruhe, Germany) using CuKα radiation. The
measurement conditions were as follows: tube voltage of 40 kV, tube current of 40 mA, step
scan mode with a step size of 2θ = 0.02°, and counting time of 0.1 s per step.
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2.5. Determination of Chlorantraniliprole Content
The content of the active ingredient was determined by high performance liquid
chromatography (HPLC) (WAT035876, Waters Co., Milford, MA, USA) using a C18 column (5 um, 4.6
mm 250 mm, Shiseido, Tokyo, Japan) at room temperature. The mobile phase was composed
of acetonitrile and water (60:40). The flow rate was 1.0 ml/min, and the UV detector
wavelength was 254 nm. The Milli-Q water (18.2 MO.cm, TOC 4 ppb) was used for the
preparation of all the solutions in this measurement.
2.6. Suspensibility Test
The suspensibility was tested by the similar method taken in previous study [
]. The 10.4167 g
powder of the 2.5% chlorantraniliprole solid nanodispersion was added slowly to a beaker
containing 50 ml standard hard water (30 ± 1°C) and swirled by hand in a circular motion at a rate
of about 120 times per minute for 2 minutes. The suspension was transferred to a 250-ml
measuring cylinder after placing it in a water bath at 30 ± 1°C for 13 minutes. Then 200 ml
standard hard water was used to rinse the beaker and fill the cylinder to scale. Subsequently, the
measuring cylinder was stoppered and inverted 30 times by hand, and kept standing in the
30 ± 1°C water bath for 30 minutes. After removing the top 225 ml of the dispersion, the
pesticide contents of the original suspension and the remaining 25 ml of the dispersion were
measured by HPLC. In the control experiments, 0.7163 g WGs (ALTACOR and JIATENG) and
1.2563 g SC were weighed and the measurements followed the same steps as described above.
2.7. Wettability Test
The 100 ml standard hard water was added into a 250-ml beaker which was placed in a water
bath at 25 ± 1°C. When the temperature of the standard hard water reached 25°C, 5.000 g
sample was poured on the water surface at once. Immediately, time was recorded with a stopwatch
until the powder was entirely wetted by water. The average value of three tests was adopted.
The 2.5% chlorantraniliprole solid nanodispersion, the commercial WGs (ALTACOR and
JIATENG) and GR were all tested under the same condition.
2.8. Retention Test
Firstly, the 2.5% chlorantraniliprole solid nanodispersion and commercial SC were diluted into
aqueous dispersions containing 0.015 mg/ml active ingredient. Then the weight of each rice
(Oryza sativa L.) leaf was weighed using an electronic balance (ME204E, METTLER TOLEDO,
Zurich, Switzerland) and its area was measured by a leaf area meter (Yaxin-1241, Beijing Yaxin
Science Instrument Technology Co., Ltd., Beijing, China). Afterwards, the leaves were fully
immersed in the above dispersions and pure water which was as a control test. After 10 s, each
leaf was pulled out when there were no droplets falling from the surface. Finally, it was placed
in a weighed culture dish to get the weight of the leaf after immersion. The weights and areas of
the leaves were accurate to 0.1 mg and 0.1 cm2, respectively. The average value of three tests
Bioassays were performed using the leaf-dip method as described in the research of the
lambda-cyhalothrin solid nanodispersion [
]. In the biological activity assay of the
chlorantraniliprole technical (TC), the TC powder was added to dimethyl sulfoxide and treated with an
ultrasonic machine (KQ-500DE, Kunshan Ultrasonic Instruments Co., Ltd., Jiangsu, China)
for 5 minutes to acquire a 10 g/l solution. Then it was diluted with Triton X-100 aqueous
5 / 16
solution to obtain the dispersions with different chlorantraniliprole concentrations and 0.05%
Triton X-100. For the chlorantraniliprole solid nanodispersion and SC, the samples were
directly diluted with pure water to different concentrations followed by 5 minutes of
ultrasound. Subsequently, rape (Brassica campestris L.) leaves were immersed in the above
dispersions for 10 s. Afterwards, the leaves were air-dried and placed in a culture dish with a filter
paper. Ten second-instar diamondback moth (Plutella xylostella L.) larvae were introduced
into each dish, and three replications were carried out. The control test in which leaves were
only treated with 0.05% Triton X-100 solution was also conducted for comparison. Mortality
was assessed after treatment for 48 h. Concentration–mortality data were analyzed using DPS
8.1 (Refine Information Technology Co., Ltd., Hangzhou, China).
2.10. Statistical Analysis
The data were analyzed by one-way analysis of variance (ANOVA) and Duncan’s multiple
range test. Statistical analysis was performed with the software package SPSS and a value of
P < 0.05 was deemed to be statistically significant.
3 Results and Discussion
The preparation of the chlorantraniliprole solid nanodispersion involved two sequential
processes: producing the chlorantraniliprole nanosuspension by HPH and solidifying the aqueous
dispersion by adding water-soluble carrier and removing water. As demonstrated previously
], the particle size and distribution of the pre-prepared nanosuspension have a crucial impact
on the properties of the final solid nanodispersion, thus the composition and preparation
parameters of the nanosuspension have been investigated in detail using the particle size and
PDI as evaluation indices.
3.1. Surfactant Screening and Composition Optimization
Surfactant as an indispensable part of pesticide formulation remarkably affects the formulation
performance. In this research, four anionic (SDS, SL, MRES and polycarboxylate), three
nonionic (PVP K90, HPMC and PGME) and one cationic (CTAB) surfactants were compared.
The nanosuspensions containing 1% (w/w) chlorantraniliprole and 0.2% (w/w) single
surfactant were produced by homogenization at 300 bar, 600 bar, 900 bar, 1200 bar and 900 bar for
10 cycles at each pressure. The pesticide particles suspended in the dispersions through the
stabilizing effect of the surfactants. The amphiphilic surfactants adsorbed on the pesticide surface
by hydrophobic interactions while leaving the hydrophilic end stretching outside. This
structure could enhance the wettability and dispersibility of the poorly water-soluble pesticide in
water. Table 1 shows the surfactant effect on the particle size and dispersibility of the
nanosuspensions. Among the eight surfactants, MRES and polycarboxylate reduced the mean size and
D90 of the particles to less than 29 nm and 76 nm, respectively. The -SO3- group on the MRES
molecular skeleton and -COO- group of polycarboxylate may interact with -NH group of
chlorantraniliprole molecule through hydrogen bonds and make the surfactants adsorb on the
pesticide surface [
]. Furthermore, the Van der Waals force between chlorantraniliprole
and surfactants may also enhance their intermolecular interactions. The anionic surfactants on
the pesticide surface made the particles negatively charged and repel each other to prevent the
formation of large particles. Meanwhile, the hydrophobic chains of MRES and polycarboxylate
could further induce steric effect against aggregation. Considering both MRES and
polycarboxylate were capable of significantly reducing the mean size (P < 0.05) and distribution of the
particles, the two surfactants were mixed and the appropriate proportion between them was
6 / 16
a SDS: 1-dodecanesulfonic acid sodium salt; SL: sodium ligninsulfonate; MRES: maleic rosin-polyoxypropylene-polyoxyethylene ether sulfonate; PVP K90:
polyvinylpyrrolidone K90; HPMC: hydroxypropyl methylcellulose; PGME: polyethylene glycol mono-4-nonylphenyl ether; CTAB:
b D90: particle size expressed by the 90% diameter percentile.
c PDI: polydispersity index.
d S.D.: standard deviation of three measurements.
Different letters at each data indicate significant differences according to Duncan’s multiple range test at P < 0.05.
Using MRES and polycarboxylate as composite surfactants, the particle sizes and PDIs of
the chlorantraniliprole nanosuspensions with different ratios of MRES to polycarboxylate were
shown in Table 2. In contrast with the nanosuspensions stabilized by single surfactant, the
mean size and D90 of the nanosuspension containing composite surfactants decreased even
reducing the relative content of surfactant to pesticide. While the two surfactants adsorbing on
the pesticide surface, both electrostatic repulsion and steric stabilization effects contributed to
the nanosuspension stability by preventing particles from aggregation. The 1:1 (w/w) ratio of
MRES to polycarboxylate was chosen in the following preparation of the chlorantraniliprole
The content of surfactants affects their adsorption amount on the surface of poorly
watersoluble pesticide, and further influences the dispersibility and stability of the formulation. Fig 2
shows the effect of surfactant-to-pesticide ratio on the particle size and distribution of the
chlorantraniliprole nanosuspensions. When the surfactant was not enough to provide efficient
electrostatic repulsion and steric hindrance, the neighboring nanoparticles may approach and
a MRES: maleic rosin-polyoxypropylene-polyoxyethylene ether sulfonate.
b The nanosuspensions containing 5% (w/w) chlorantraniliprole and 0.25% (w/w) surfactants were prepared by homogenization at 300 bar, 600 bar, 900 bar,
1200 bar and 900 bar for 10 cycles at each pressure.
c D90: particle size expressed by the 90% diameter percentile.
d PDI: polydispersity index.
e S.D.: standard deviation of three measurements.
Different letters at each data indicate significant differences according to Duncan’s multiple range test at P < 0.05.
7 / 16
Fig 2. The particle size and dispersibility of the nanosuspensions containing 5% (w/w)
chlorantraniliprole with different surfactant-to-pesticide ratios. D90: particle size expressed by the 90%
diameter percentile; PDI: polydispersity index. Different letters at each data indicate significant differences
according to Duncan’s multiple range test at P < 0.05.
aggregate. Indeed, as the ratio increased from 1:80 to 1:20, the mean size and D90 of the
pesticide nanoparticles decreased gradually. However, the particle size and PDI changed little and
even appeared a slight increase when the ratio was larger than 1:20. The possible reason was
that more surfactants made the interface layer of particles become thicker [
]. In addition, the
large amount of surfactants would cause greater food safety and environmental issues [
Therefore, the 1:20 surfactant-to-pesticide ratio was determined for the preparation of the
solid nanodispersions. The optimized surfactant content was much lower than that in most
microemulsions and solid microemulsions [
3.2. Homogenization Process Effect
Homogenization condition has a significant impact on the particle size and structure during
the preparation of nanosuspensions [
]. In this investigation, to avoid a blockage of the
homogenization gap caused by large particles [
], the mixed dispersion of pesticide and
surfactants was first sheared to reduce the particle size to about 600 nm before passing it into the
homogenizer. The effects of both homogenization mode and pressure on the particle size and
dispersibility of the chlorantraniliprole nanosuspensions have been explored. As the
homogenization pressure increased, the enhanced shear and cavitation forces made big particles split
into small ones, consistent with the result in Fig 3a. The reduction of the mean size of particles
was obvious from 300 bar to the first 900 bar (P < 0.05), but the change became slight during
Fig 3. The particle size and dispersibility of the nanosuspensions containing 5% (w/w)
chlorantraniliprole prepared in (a) variable pressure mode and (b) constant pressure mode. D90:
particle size expressed by the 90% diameter percentile; PDI: polydispersity index. Different letters at each
data indicate significant differences according to Duncan’s multiple range test at P < 0.05.
8 / 16
the further homogenization process. It has been reported that input of more energy may lead
to “over-processing” and induce particle growth because it accelerates particle movement and
causes aggregation [
]. Similar phenomenon also exists in the milling process [
Therefore, in order to substantially improve the dispersibility of the nanosuspension while
avoiding particle aggregation, a lower 900 bar was adopted following 1200 bar to further
enhance the system stability. The above result demonstrates that homogenization pressure
could effectively regulate the size and distribution of the nanoparticles. The comparative study
was conducted in the constant pressure mode at 1200 bar. As shown in Fig 3b, the mean size
and D90 of the particles decreased progressively with increasing cycle number. The increase of
cycle number provided sufficient time for the surfactants to adsorb onto the pesticide surface
and breaking large particles. By comparison, the final particle size in the variable pressure
mode was equal to that after passing 35 cycles at 1200 bar. Considering the energy
consumption problem, the variable pressure mode was chosen.
The water-based suspensions are thermodynamically unstable systems that tend to break
down over time due to a variety of physicochemical mechanisms, for example, gravitational
separation, flocculation and Ostwald ripening [
]. In order to improve the stability and prolong
shelf life of the formulation, the chlorantraniliprole nanosuspensions were transformed into
solid nanodispersions by lyophilization. During this process, sucrose was added before
freezedrying. Here, sucrose as water-soluble carrier can not only act as antifreeze agent to protect
dispersion from freezing and desiccation impairment, but also accelerate redispersion of the solid
nanodispersions as proved by other systems [
]. Furthermore, it can also improve the
suspensibility and stability of the re-dispersed dispersion by increasing its viscosity [
After getting the identical nanosuspensions by the same preparation process, the adding
amount of sucrose was regulated to prepare the solid nanodispersions with different pesticide
contents. The content of the active ingredient gradually increased as the proportion of sucrose
in the composition decreased. For the 2.5%, 7.5%, 22.5% and 67.5% solid nanodispersions, the
formulations consisted of chlorantraniliprole, composite surfactants of MRES and
polycarboxylate and sucrose. By contrast, in order to maximize the pesticide content, the nanosuspension
was directly lyophilized without adding sucrose to get the 91.5% solid nanodispersion. In this
condition, the formulation was only composed of chlorantraniliprole and surfactants. As
shown in Fig 4, the mean size of nanoparticles became larger as increasing pesticide content
but all less than 75 nm. Similar phenomenon has been observed in nanosuspension systems
Fig 4. The particle size and dispersibility of five chlorantraniliprole solid nanodispersions with
different pesticide contents. D90: particle size expressed by the 90% diameter percentile; PDI:
polydispersity index. Different letters at each data indicate significant differences according to Duncan’s
multiple range test at P < 0.05.
9 / 16
and clarified as due to achieving a more stable state by coalescence [
]. It is noteworthy that
the chlorantraniliprole content in the solid nanodispersion could reach up to 91.5%, while
keeping the mean particle size, D90 and PDI at 68 nm, 191 nm and 0.24, respectively. This
high concentration nanoformulation maximized the active ingredient and minimized the
surfactants, solvent and carrier. It could dramatically reduce the production cost and improve
safety, environmental friendliness and utilization convenience of the product. Moreover, it is
an ideal candidate for oil formulations in the application of ultra-low volume and aerial spray.
3.3. Characterization and Evaluation of the Solid Nanodispersion
3.3.1. Size and Morphology. The solid nanodispersion containing 2.5% (w/w)
chlorantraniliprole was taken as an example to be evaluated in detail. The mean size, D90 and PDI of the
nanoparticles measured by DLS were 29 ± 1 nm, 91 ± 6 nm and 0.26 ± 0.01, respectively (Fig
5a). The slight agglomeration of particles during lyophilization has also been reported in other
]. As observed from the SEM and TEM images (Fig 5b and 5c), the
nanoparticle size was mainly in the range of 25 nm to 135 nm, agreed with the result of DLS. During
the homogenization process, the shear, cavitation and collision forces were asymmetrically
applied to the particles and resulted in the irregularity of the particle shape.
3.3.2. Zeta Potential and pH. Zeta potential is a typical index to evaluate the surface
charge property of particles and the physical stability of water-based formulations [
zeta potential and pH of the re-dispersed solid nanodispersion were– 22 mV and 7.4,
respectively. The negative zeta value demonstrates that the anionic surfactants adsorbed on the
pesticide surface and made the particles present electronegativity. In general, absolute zeta potential
values higher than 30 mV predict a strong long-term stability of a suspension. However, a
suspension with lower zeta potential can also exhibit excellent stability when the surfactants
provide steric stabilization in addition to electrostatic repulsion [
]. The adsorption layer of
the chlorantraniliprole nanoparticles consisted of anionic polymers MRES and polycarboxylate
which both acted as electrostatic repulsion and steric stabilizers. This may lead to a shift of the
shear plane to a larger distance from the particle surface, and thus, to a reduction in the
measured potential [
]. In addition, the pH close to neutral condition was conducive to
avoiding the decomposition of active ingredient.
Fig 5. The size and morphology of the chlorantraniliprole nanoparticles. (a) Particle size measured by
DLS; (b) SEM image with magnification of 15000; (c) TEM image with magnification of 25000. Size (d.
nm): diameter size of the nanoparticles.
10 / 16
3.3.3. Crystallinity. As shown in Fig 6, XRD pattern of the solid nanodispersion presented
a certain degree of amorphous characteristic compared to the pure chlorantraniliprole
nanocrystal, owing to the amorphous surfactants covering the pesticide surface. The intense peaks
of the solid nanodispersion mainly resulted from sucrose crystal which accounted for the
largest proportion in the formulation composition. As reported, HPH can induce structural change
of materials [
]. The characteristic peaks of pure pesticide at 8.7°, 10.0°, 17.3° and 30.2°
observed in the pattern indicates the preservation of pesticide crystal structure. The crystalline
state was stable during storage and the amorphous component could promote the dissolution
of poorly water-soluble compound [
]. The coexistence of crystalline and amorphous
structures will play an important role in improving storage stability and water solubility of the
chlorantraniliprole solid nanodispersion.
3.3.4. Suspensibility. Suspensibility is an important indicator of the re-dispersibility of
solid formulations and the kinetic stability of suspensions. The value was measured according
to CIPAC MT 184 and calculated by the following equation:
Suspensibilityð%Þ ¼ 9
Here, m1 (mg) and m2 (mg) are the pesticide contents of the original suspension and the left
25-ml dispersion at the bottom, respectively. As reported in the literatures, the suspensibilities
of bacillus marinus WP and fluopicolide pyraclostrobin WG after formulation optimization
were 73.13% and 90.56% [
]. In this research, the suspension properties of the
chlorantraniliprole solid nanodispersion and three commercial products were compared under the same
condition. The suspensibilities of the chlorantraniliprole SC, ALTACOR WG and JIATENG
WG were 96.63%, 93.60% and 86.72%, respectively. By contrast, the 97.32% suspensibility of
the solid nanodispersion indicated that the re-dispersibility and stability of the
nanoformulation were further improved. There is an inverse relationship between suspensibility and particle
size , because the relatively small particle size means that Brownian motion may dominate
the gravitational force [
]. In addition, sucrose as a thickener increased solution viscosity and
decreased particle sedimentation velocity. Therefore, the excellent suspension characteristic of
the solid nanodispersion could attribute to the small size effect and formulation composition.
3.3.5. Wettability and Retention. Wettability and retention influence the pesticide
efficacy by affecting spread and adhesion of aqueous dispersions on leaves after spraying. The
wettability was evaluated according to CIPAC MT 53 and the wetting times of the commercial
chlorantraniliprole GR, ALTACOR WG and JIATENG WG were 373 s, 84 s and 65 s,
respectively. In contrast, the solid nanodispersion could be wetted by water within 13 s which was
only one fifth of that for conventional formulations. The retention (Rm, mg/cm2) was measured
according to the literature [
] and calculated by the following equation:
Here, W0 (mg) and W1 (mg) are the weights of the hydrophobic rice (Oryza sativa L.) leaf
before and after immersing in aqueous dispersions, S (cm2) is the area of the rice (Oryza sativa
L.) leaf. As shown in Table 3, the retention of the chlorantraniliprole solid nanodispersion was
1.5 and 3 times that of commercial SC and pure water, respectively. The above results
demonstrate that the size reduction, enlarging specific surface area of particles and their contact
area with leaves, is indeed beneficial to shortening wetting time and increasing retention of
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Fig 6. XRD patterns of the chlorantraniliprole solid nanodispersion and pure components in the formulation. MRES: maleic
rosin-polyoxypropylene-polyoxyethylene ether sulfonate.
3.3.6. Storage Stability. The stability of the solid nanodispersion was tested according to
CIPAC MT 46 and GB/T 19136–2003. As shown in Fig 7a, within five days, the mean particle
size increased from 29 nm to 62 nm during storage at 54°C, but kept constant up to 14 days.
The particle growth during storage also exists in microemulsions and nanosuspensions because
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Fig 7. Stability of the chlorantraniliprole solid nanodispersion at (a) 54°C and (b) 25°C. D90: particle
size expressed by the 90% diameter percentile; PDI: polydispersity index. Different letters at each data
indicate significant differences according to Duncan’s multiple range test at P < 0.05.
of Ostwald ripening [
]. By contrast, the size and PDI of the nanoparticles remained
almost unchanged during storage at 25°C, indicating excellent storage stability (Fig 7b). The
amorphous characteristic of the nanoparticles caused slight aggregation at high temperature.
However, the crystalline component and narrow size distribution could effectively prevent
particle coalescent and recrystallization at room temperature, improving the stability of the solid
3.3.7. Biological Activity. The bioassay result of the chlorantraniliprole solid
nanodispersion to diamondback moths (Plutella xylostella L.) was compared with TC and SC as shown in
Table 4. The toxicity of the solid nanodispersion was 3.3 and 2.8 times that of TC and SC,
respectively. It has been reported that the bioavailability of nanoemulsion is higher than that of
conventional emulsion because of its small particle size and high surface-to-volume ratio [
Therefore, the enhanced biological activity of the solid nanodispersion here can be attributed
to the improvement of the formulation performance in dispersibility, wettability and retention
caused by small size effect. The formulation with high bioavailability could substantially reduce
usage, decrease residue and improve environmental friendliness of pesticide.
In this research, the solid nanodispersions of poorly water-soluble chlorantraniliprole with
high melting point were prepared by high pressure homogenization combined with
lyophilization. The formulation avoided organic solvent, substantially reduced surfactant amount and
could increase pesticide content to 91.5%. The solid nanodispersion with mean particle size of
29 nm presented improved formulation characteristics in dispersibility, wettability, stability
and bioavailability compared to the conventional pesticide formulations. Therefore, the
application of the solid nanodispersion in crop protection has great potential for reducing residues
in food and environmental pollution of pesticide.
a LC 50: median lethal concentration.
b TC: technical.
c SC: aqueous suspension concentrate.
LC 50a (μg/mL)
95% confidence limit
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Conceived and designed the experiments: BC HC.
Performed the experiments: BC LF CW DY.
Analyzed the data: BC.
Contributed reagents/materials/analysis tools: MY YW CS XZ.
Wrote the paper: BC ZZ HC.
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