nanomaterials

Article
Dual-Functionalized Pesticide Nanocapsule Delivery
System with Improved Spreading Behavior and
Enhanced Bioactivity
Jianxia Cui † , Changjiao Sun † , Anqi Wang, Yan Wang *, Huaxin Zhu, Yue Shen , Ningjun Li,
Xiang Zhao, Bo Cui, Chong Wang, Fei Gao, Zhanghua Zeng and Haixin Cui *
Institute of Environment and Sustainable Development in Agriculture, Chinese Academy of Agricultural
Sciences, Beijing 100081, China; [email protected] (J.C.); [email protected] (C.S.);
[email protected] (A.W.); [email protected] (H.Z.); [email protected] (Y.S.);
[email protected] (N.L.); [email protected] (X.Z.); [email protected] (B.C.); [email protected] (C.W.);
[email protected] (F.G.); [email protected] (Z.Z.)
* Correspondence: [email protected] (Y.W.); [email protected] (H.C.);
Tel.: +86-10-82105997 (Y.W.); +86-10-82106013 (H.C.)
† These authors contributed equally to this work.




Received: 6 January 2020; Accepted: 23 January 2020; Published: 27 January 2020


Abstract: The prevention and control of pests and diseases are becoming increasingly difficult
owing to extensive pesticide resistance. The synergistic use of pesticides for disease control is
an effective way of slowing pesticide resistance, reducing the number of pesticide applications,
and protecting the environment. In this study, a dual-functionalized pesticide nanocapsule delivery
system loaded with two active ingredients (AIs)—validamycin and thifluzamide—was developed
to prevent and control rice sheath blight; the nanocapsule system was based on a water–oil–water
double emulsion method combined with high-pressure homogenization technology. Our results
showed that the dual-functionalized pesticide nanocapsules were monodisperse spheres with a mean
particle size of ~260 nm and had good storage stability. Compared with commercial formulations,
the dual-functionalized pesticide nanocapsules exhibited good foliar spread owing to their small
size, which is beneficial for reducing the loss of pesticides on the leaves. The 50% median effect
concentration and synergistic ratio against Rhizoctonia solani of the dual-functionalized pesticide
nanocapsules and commercial formulation were 0.0082 and 0.0350 µg/mL, and 2.088 and 0.917,
respectively. These findings indicate that the bioactivity of the dual-functionalized system was
significantly better than that of the commercial formulations and that the dual-functionalized system
demonstrated a clear synergistic effect between the two AIs. The system presented here is simple,
fast, and capable of dual-pesticide loading with significant synergistic effects. Our findings could
help to facilitate the improvement of pesticides efficiency and the slowing of pesticide resistance.

Keywords: validamycin; thifluzamide; dual-functionalized pesticide nanocapsules; storage stability;

foliar spread; bioactivity; synergistic effect

1. Introduction
Sheath blight is one of the most harmful diseases in rice (Oryza sativa L.) worldwide, and is the
most prevalent among the three major rice diseases in China [1,2]. For several decades, the control of
rice sheath blight has relied primarily on the use of validamycin. However, due to the emergence of
pesticide resistance, the control efficiency of validamycin has declined in recent years [3,4]. In addition,
when conventional pesticide formulations are used to control rice sheath blight, their active ingredients
(AIs) can be reduced as a result of spray drift, run-off, and degradation during field application [5–10].

Nanomaterials 2020, 10, 220; doi:10.3390/nano10020220 www.mdpi.com/journal/nanomaterials

Nanomaterials 2020, 10, 220 2 of 13

Owing to their low efficiency, the extensive use of conventional pesticide formulations has had
deleterious effects such as environmental pollution, threats to non-target organisms, and pesticide
resistance; these cumulative effects significantly aggravate food safety issues, posing a significant
threats to human health [11–15].
The modification of existing pesticides and the development of novel pesticides with high control
efficiency and low toxicity is essential to slow the progression of pesticide resistance [16]. However,
the development of new AI is becoming increasingly difficult, making it more desirable to formulate
complex pesticide formulations through precise selection and targeted combination of AIs based on
their synergistic effects [17]. This provides an effective approach for improving the utilization efficiency
of existing pesticides, delaying the occurrence of pathogen resistance, broadening the spectrum of
disease control, and prolonging the service life of pesticide formulations. However, the complex
formulations available on the market are simply mixtures of existing pesticide formulations. Although
these formulations can slow the occurrence of pesticide resistance to a certain extent, unsolved problems
remain, such as the requirement for large amounts of organic solvents, drift, and poor dispersity in
water [18,19]. Furthermore, conventional pesticide formulations always rapidly fall below the effective
concentration level due to hydrolysis, photolysis or microbial degradation [19]. A delivery system
could effectively prevent the premature degradation of pesticides, achieve continuous and stable
release of AIs, and maintain a predetermined minimum effective level of pesticides for a suitable time
period [20].
Encapsulation technology has attracted emerging interest. By encapsulating liquid or solid
pesticides into the shell material, capsules could protect AIs from the degradation caused by
environmental factors, which provides a new strategy for improving the utilization rate of
pesticides [21–24]. Nanocapsules can also reduce the loss of pesticides to non-target environments,
achieve sustainable release of AIs, and maintain effective control concentrations over extended periods
of time [8,25–29]. However, most of the available pesticide capsules are on the micron scale [30].
By comparison, nanocapsules demonstrate clear advantages for improving the foliar deposition and
spread of pesticides and enhancing their bioactivity due to their small-size and large specific surface
area [31–35]. The preparation of nanocapsules requires strict conditions [36–39], however, research on
dual-pesticide nanocapsules is relatively scarce.
Thifluzamide is a novel and highly effective systemic fungicide that is used for the control of
rice sheath blight [40–42]. In this study, thifluzamide and validamycin were selected as a synergistic
combination. Dual-functionalized pesticide nanocapsules were prepared based on the distinct physical
and chemical properties of the selected pesticides. Our findings demonstrate that the ratio of the
pesticides, the physicochemical properties, and the morphology, particle size, and structure of the
dual-functionalized pesticide nanocapsule delivery system can be adjusted to improve the field control
efficiency of the pesticides. This control of pesticide application slowed the occurrence of pathogen
resistance, broadened the microbicidal spectrum, and reduced the cost of pesticide application.

2. Materials and Methods

2.1. Materials
Validamycin A (60%) and thifluzamide (95%) were purchased from Bailexin Biotech. Co., Ltd.
(Beijing, China). A validamycin standard was obtained from Leboward Tech. Co., Ltd. (Beijing, China)
and a thifluzamide standard was supplied by Bailinwei Tech. Co., Ltd. (Beijing, China). Validamycin
wettable powder (VWP) and aqueous solution (VAS) were supplied by Huifeng Biosciences Co.,
Ltd. (Tonglu, China) and Lvchuan Biotech. Industry Co. Ltd. (Fuzhou, China), respectively.
The thifluzamide suspension concentrate (TSC) was manufactured by Limin Agrochemical Co., Ltd.
(Yancheng, China), and the validamycin/thifluzamide suspension concentrate (VTSC) was supplied by
Nannong Pesticide Technology Development Co., Ltd. (Nanjing, China).
Nanomaterials 2020, 10, 220 3 of 13

Polylactide (PLA, Mw ~ 100 KDa) was supplied by Nature Works Co. (Minnetonka, MN, USA).
Polyvinyl alcohol (PVA), 87%–90% hydrolyzed with an average Mw of 30,000–70,000, was supplied
by Sigma-Aldrich Shanghai Trading Co., Ltd. (Shanghai, China). Styryl phenol polyoxyethylene
ether (emulsifier 600#), alkylphenol formaldehyde resin polyoxyethylene ether (emulsifier 700#), and
polyoxyethylene castor oil ether (EL-40) were purchased from Cangzhou Hongyuan Agrochemical Co.,
Ltd. (Cangzhou, China). Polyoxyethylene sorbitan monooleate (Tween 80) was purchased from J&K
Chemical (Beijing, China). Maleic rosinpolyoxypropylene-polyoxyethylene ether sulfonate (MRES)
and polycarboxylate (PC) were provided by Sinvochem S&D Co., Ltd. (Jiangsu, China). Octylphenol
polyoxyethylene ether-10 (OP-10) was purchased from Jinyue Biotech. Co., Ltd. (Beijing, China).
Sodium dodecyl sulfate (SDS) was supplied by BioMS Biotech. Co., Ltd. (Beijing, China). Castor
oil polyoxyethylene ether-125 (BY-125) was obtained from Jiangsu Zhongshan Chemical Co., Ltd.
(Nanjing, China).
Analytical-grade dichloromethane and chloroform were obtained from Beijing Chemical Works
(Beijing, China). Acetonitrile and methanol (high-performance liquid chromatography (HPLC) grade)
were purchased from Fisher (Shanghai, China). The water used in all experiments was purified using a
Milli-Q water purification system (Dubuque, IA, USA).

2.2. Methods

2.2.1. Preparation of Dual-Functionalized Pesticide Nanocapsules

The dual-functionalized pesticide nanocapsule delivery system was prepared using a
water–oil–water (W/O/W) double emulsion method combined with high pressure homogenization
technology. Briefly, 2.5 g of validamycin was dissolved in 15 mL of water to form an aqueous solution
as the internal water phase. Next, 0.5 g of thifluzamide was dissolved in 60 mL of dichloromethane,
followed by the addition of 3 g of PLA (capsule wall material) to form the oil phase. The external
water phase was obtained by dissolving 4 g of surfactant in 300 mL of water. The internal water phase
was dispersed in the oil phase using an ultrasonic homogenizer (JY92-IIN, Sientz, Ningbo, China)
at 585 W to form a primary W/O emulsion, which was then added to the external water phase and
sheared for 5 min at 19,000 rpm to prepare a coarse W/O/W double emulsion. To reduce the size of
the emulsion particles, the coarse emulsion was transferred to a high-pressure homogenizer (ATS,
AH-100D, Engineer Inc., Brampton, Canada) and treated three times under successive pressures of 300,
600, and 900 kPa to yield a fine double emulsion.
The obtained emulsion was mechanically stirred at 800 rpm for 20 h at room temperature using
an electric stirrer (IKA, EUROSTAR 60, Staufen, Germany) to allow for solidification into capsules and
eliminate excess organic solvents. The nanocapsules were collected by centrifugation at 10,000 rpm for
10 min at room temperature (Thermo SCIENTIFIC, ST 16R, Pittsburgh, PA, USA), and the precipitate
was resuspended in deionized water. This process was repeated three times to remove excess impurities.
Then, the precipitate was collected and frozen in an ultra-low-temperature freezer (Haier, DW-86W100,
Qingdao, China). Excess water was removed using a freeze dryer (FD-81, ETELA, Tokyo, Japan).

2.2.2. Particle Size and Morphological Characterization of Dual-Functionalized

Pesticide Nanocapsules
A small portion of the solid powder was diluted in deionized water to obtain a transparent
suspension. The mean particle size (MPS) and polydispersity index (PDI) of the nanocapsules in the
transparent solution were measured by dynamic light scattering (DLS Zetasizer Nano ZS90, Malvern
Instruments, Worcestershire, UK). Each sample was measured in parallel three times, and the mean
values and standard deviation were calculated.
A scanning electron microscope (SEM, HITACHI, Su-8010, Tokyo, Japan) was used to characterize
the morphology of the nanocapsules. An aliquot of the resuspended nanocapsules was dropped onto
the surface of a cleaned silicon water. The SEM images were acquired at an acceleration voltage of 5 kV.
Nanomaterials 2020, 10, 220 4 of 13

A transmission electron microscope (TEM, HITACHI, HT7700, Tokyo, Japan) was used to characterize
the internal structure of the prepared nanocapsules. An appropriate volume of the transparent solution
was dropped onto 300-mesh copper grid coated with a carbon film. TEM images were acquired at an
acceleration voltage of 80 kV.

2.2.3. Determination of Dual-Functionalized Pesticide Nanocapsules’ Loading Efficiencies

The loading efficiency of the two AIs into the nanocapsules was determined by destruction of the
nanocapsules. An appropriate aliquot sample was accurately weighed by an ultramicro analytical
balance (METTLER TOLEDO, Zurich, Switzerland) and completely dissolved in chloroform to break
the capsule wall. Excess solvent was removed by distillation under reduced pressure using a rotary
evaporator (RE100-Pro, SCILOGEX, Rocky Hill, CT, USA). We then used 90% methanol to extract
the pesticides before the dissolved sample of the two AIs was passed through a 0.22 µm membrane
filter. The pesticide concentrations in the filtrate were determined by HPLC (1260 Infinity, Agilent
Technologies, Palo Alto, CA, USA) using a C18 column (5 µm, 4.6 mm × 150 mm, Agilent Technologies;
Santa Clara, CA, USA) at room temperature. The mobile phase for validamycin was 0.005 mol/L
disodium hydrogen phosphate buffer solution (pH = 7.0), with a flow rate of 0.8 mL/min and a detection
wavelength of 210 nm; the mobile phase for thiafuramide was acetonitrile/water (70:30) with a flow
rate of 1 mL/min and a detection wavelength of 225 nm.
The loading efficiency of the nanocapsules was calculated as follows:
Pesticide loading rate (%) = effective mass of pesticide in nanocapsules/total mass of nanocapsules
× 100.

2.2.4. Stability Evaluation of Dual-Functionalized Pesticide Nanocapsules

The stability of the dual-functionalized pesticide nanocapsules was evaluated according to
the General Principles of Pesticide Storage Stability Test at Normal Temperature (NY/T 1427–2016),
Pesticide Thermal Storage Stability Test Method (GB/T 19136–2003), and Pesticide Cold Storage Stability
Test Method (GB/T 19137–2003). Specifically, three parallel samples of the transparent solution were
transferred into ground–glass tubes and sealed and stored separately at 0 ± 2 ◦ C for 7 days and
25 ± 2 ◦ C and 54 ± 2 ◦ C for 14 days. Samples were removed at specific time intervals for stability
evaluation based on MPS and PDI measurements.

2.2.5. Foliar Spread and Contact Angle Evaluation of Dual-Functionalized Pesticide Nanocapsules
Cucumber (Cucumis sativus L.)—the chosen plant model—was grown in an artificial climate
incubator (430D, Ningbo Jiangnan Instrument, Ningbo, China). Nanocapsules with a concentration
of 200 µg/mL were sprayed onto the surface of the cucumber leaves and allowed to air-dry at room
temperature. Control groups were treated with commercially available VTSC, TSC, VAS, and VWP.
The dispersion of different samples on the foliage was observed using an environmental scanning
electron microscope (ESEM; Quanta FEG 250, Houston, TX, USA).
To evaluate the contact angle, 7 µL of 200 µg/mL nanocapsule solution was dropped onto the
surface of fresh leaves at room temperature using a microliter syringe. Images of each droplet were
immediately captured using a contact angle goniometer (JC2000D2M; Zhongchen Digital Technology
Equipment Co., Ltd., Shanghai, China). Each trial was repeated five times.

2.2.6. Bioactivity Evaluation of Dual-Functionalized Pesticide Nanocapsules

Rhizoctonia solani, the pathogen responsible for rice sheath blight, was selected as a model fungus
to evaluate the bioactivity of the nanocapsules. Commercial VTSC, VWP, and TSC were used as
controls and sterile deionized water was used as the blank control. Briefly, a 5-mm-diameter punch was
used to cut mycelium plugs from an activated culture of R. solani that grew uniformly. The plugs were
inoculated onto the center of 90 mm-diameter potato dextrose agar plates. The plates were incubated
at 25 ◦ C for 36 h, and three parallel independent trials were performed for each concentration. Colony
Nanomaterials 2020, 10, 220 5 of 13

diameter was measured using the cross method and statistically analyzed. Growth inhibition rate and
synergistic ratio (SR) were calculated. In addition, the toxicity regression equation and median effect
concentration (EC50 ) were analyzed using Data Processing System (DPS, v7.05) [43].
The growth inhibition rate was calculated as follows:
Nanomaterials 2020, 9, x FOR PEER REVIEW 5 of 13

performed for each concentration. = (Dc −diameter

I%Colony − Dmeasured
Dt )/(Dcwas d ) × 100% using the cross method and (1)
statistically analyzed. Growth inhibition rate and synergistic ratio (SR) were calculated. In addition,
wherethe
I%toxicity
is the growth inhibition
regression equation rate of R. solani;
and median effectD c and Dt are(EC
concentration the50)growth diameter
were analyzed of R.
using solani in the
Data
blankProcessing
control andSystem
the treatment group,
(DPS, v7.05) [43].respectively; and Dd is the diameter of the mycelium plug (5 mm).
The growth inhibition
The interaction rate was
of the mixed calculated
pesticides as follows:
was evaluated according to the Wadley method using the
following equations: I % = (D c − Dt )/ (D c − Dd) ×100％ (1)
where I% is the growth inhibition rate of R. solani; Dc and Dt are the growth diameter of R. solani in
the blank control andEC (theoretical
the50treatment value)
group, = (a + b)/(a/EC
respectively; and Dd is + diameter
50athe b/EC50b )of the mycelium (2)
plug (5 mm).
The interaction of the = EC50
SR mixed (theoretical
pesticides value)/EC
was evaluated 50 (actual
according to value)
the Wadley method using (3)
the following equations:
where a and b are the proportions of the two pesticides in the mixture. The effect of the mixture was
analyzed based on the SREC 50 (theoretical value) = (a + b)/(a/EC 50a + b/EC 50b )
value: SR ≤ 0.5 indicates an antagonistic effect; SR = 0.5–1.5(2) indicates an
additive effect; and SR ≥ 1.5
SRindicates a synergistic
= EC 50 (theoretical effect
value)/ EC 50between the two pesticides [44].
(actual value) (3)
where a and b are the proportions of the two pesticides in the mixture. The effect of the mixture was
2.2.7. analyzed
Statistical Analysis
based on the SR value: SR ≤ 0.5 indicates an antagonistic effect; SR = 0.5–1.5 indicates an
additive effect;
Statistical and SR
analysis of≥the
1.5 indicates a synergistic
experimental data waseffectcarried
betweenout
theusing
two pesticides
DPS. The [44].
results are presented
as “average value ± standard deviation”. The multiple comparative analysis was performed using the
2.2.7. Statistical Analysis
least significant difference (LSD) method. (*) P < 0.05, was regarded a significant difference between
Statistical analysis of the experimental data was carried out using DPS. The results are
experimental groups.
presented as “average value ± standard deviation”. The multiple comparative analysis was
performed using the least significant difference (LSD) method. (*) P < 0.05, was regarded a
3. Results and Discussion
significant difference between experimental groups.

3.1. Preparation of Dual-Functionalized

3. Results and Discussion Pesticide Nanocapsules
PLA, a safe, degradable, and low-cost polymer was selected as the capsule material for this
3.1. Preparation of Dual-Functionalized Pesticide Nanocapsules
study. Multiple emulsification methods including ultrasonication, shearing, and high-pressure
PLA, a safe, degradable, and low-cost polymer was selected as the capsule material for this
homogenization, were adopted to prepare a delivery system with small particle size that was
study. Multiple emulsification methods including ultrasonication, shearing, and high-pressure
simultaneously loaded with two pesticides AIs (Figure 1). PVA and polycarboxylate were selected as
homogenization, were adopted to prepare a delivery system with small particle size that was
surfactants to stabilize
simultaneously thewith
loaded nanocapsules.
two pesticidesPVA, a non-ionic
AIs (Figure 1). PVA surfactant, formedwere
and polycarboxylate an adsorptive
selected as layer to
produce a stericto
surfactants hindrance effect
stabilize the to preventPVA,
nanocapsules. nanocapsules’ aggregation
a non-ionic surfactant, [45].anPolycarboxylate,
formed adsorptive layer toan anionic
produce
surfactant, a steric hindrance
provided sufficient effect to prevent
electrical nanocapsules’
charge to increase aggregation [45]. Polycarboxylate,
the electrostatic an
repulsion between the
anionic surfactant, provided sufficient electrical charge to increase the electrostatic
nanocapsules [46]. The simultaneous use of surfactants with different properties improved the stability repulsion
between the nanocapsules [46]. The simultaneous use of surfactants with different properties
of the dual-functionalized pesticide nanocapsules.
improved the stability of the dual-functionalized pesticide nanocapsules.

Figure
Figure 1. Schematic
1. Schematic illustration of
illustration of the
thepreparation
preparationof dual-functionalized pesticide
of dual-functionalized nanocapsules.
pesticide nanocapsules.
Nanomaterials 2020, 9, x FOR PEER REVIEW 6 of 13
Nanomaterials 2020, 9, x FOR PEER REVIEW 6 of 13
3.2. Effects of Surfactants on the Particle Size and Polydispersity of Dual-Functionalized Pesticide
Nanomaterials 2020, 10, 220 6 of 13
3.2. Effects of Surfactants on the Particle Size and Polydispersity of Dual-Functionalized Pesticide
Nanocapsules
Nanocapsules
3.2.1. SingleofSurfactants
3.2. Effects Surfactants on the Particle Size and Polydispersity of Dual-Functionalized
Pesticide
3.2.1. Nanocapsules
Single Surfactants
MPS and PDI are two important indicators used to evaluate the performance of
MPS and
dual-functionalized
3.2.1. Single PDI pesticide
Surfactants are two nanocapsules.
important indicators
Figure 2 used to evaluate
summarizes the MPSthe and
performance
PDI of theof
dual-functionalized
nanocapsules prepared pesticide nanocapsules.
with different single Figure 2 summarizes
surfactants. A total of the MPS and
10 typical PDI of were
surfactants the
MPS and PDI are two important indicators used to evaluate the performance of dual-functionalized
nanocapsules prepared
selected to explore theirwith different
effects single surfactants.
on nanocapsule synthesis,Aand
totalallof1010surfactants
typical surfactants were
had significant
pesticide nanocapsules. Figure 2 summarizes the MPS and PDI of the nanocapsules prepared with
selected
effects on tothe
explore
MPS and theirPDI
effects on nanocapsules.
of the nanocapsule synthesis,
The MPS and PDI all 10 of surfactants had significant
the nanocapsules prepared
different single surfactants. A total of 10 typical surfactants were selected to explore their effects
effects on the PVA,
with MRES, MPS andSDS,PDIandofPCthewere
nanocapsules.
all less than The300
MPSnmand
andPDI
0.3,ofrespectively,
the nanocapsules prepared
indicating good
on nanocapsule synthesis, and all 10 surfactants had significant effects on the MPS and PDI of the
with MRES,
dispersity andPVA,
smallSDS, and PC were
nanocapsule allthe
size in less than 300
presence nm and
of these four0.3, respectively, indicating good
surfactants.
nanocapsules. The MPS and PDI of the nanocapsules prepared with MRES, PVA, SDS, and PC were all
dispersity and small nanocapsule size in the presence of these four surfactants.
less than 300 nm and 0.3, respectively, indicating good dispersity and small nanocapsule size in the
presence of these four surfactants.

Figure 2. Effects of single surfactants on the mean particle size (MPS) and polydispersity index (PDI)
Figure
of Effectsof
2.2.Effects of single
the nanocapsules.
Figure single surfactants
surfactants on the
the mean
mean particle
particlesize
size(MPS)
(MPS)and
andpolydispersity
polydispersityindex
index(PDI) of
(PDI)
the
of nanocapsules.
the nanocapsules.
It was found that the emulsification effect was poor for the nanoparticles prepared with SDS,
ItIt was
which was found
found that
produced athatthe
theemulsification
coarse emulsification
unstable effect
emulsion. was
effect poor
was for the
poor
Therefore, the
for nanoparticles
the prepared
nanoparticles
morphology of with SDS,
prepared
the particles with which
SDS,
prepared
produced
which a coarse
produced
with MRES, PVA, unstable
a and
coarse emulsion.
PCunstable Therefore, the
emulsion. Therefore,
was characterized morphology of the
the morphology
by SEM. Although particles prepared
of the particles
MRES facilitated with MRES,
prepared
the formation of
PVA,MRES,
with
small and PCPVA,
particleswas characterized
withandgood
PC was by SEM. Although
characterized
dispersity, their by SEM.MRES
morphology facilitated
Although
did not MRES the facilitated
show formation
uniformly of stable
the small particles
formation of
spheres
with good
small
(Figure 3), dispersity,
particles
however theirPVA
with both
good morphology
dispersity, did not
and PC their show
morphology
supported uniformly
did notof
the formation stable
show spheres
regular (Figure
uniformly
spheres. 3), however
stable
Based spheres
on these
both
(FigurePVA
results, 3), and PC
thehowever supported
both PVA
combination the formation
and and
of PVA of
PC supportedregular spheres. Based
the formationselected
PC was preferentially on these
of regular results,
forspheres. the combination
Based on of
the preparation these
the
of PVA and
results, PCnanocapsules.
was preferentially
the combination
dual-pesticide of PVA selected
and PC for wasthepreferentially
preparation ofselected
the dual-pesticide nanocapsules.
for the preparation of the
dual-pesticide nanocapsules.

Figure 3. Scanning
Scanning electron
electron microscopy
microscopy (SEM)
(SEM) morphologies
morphologies of of the
the nanocapsules
nanocapsules (A) maleic
rosinpolyoxypropylene-polyoxyethylene
Figure
rosinpolyoxypropylene-polyoxyethylene ether
3. Scanning electron microscopyether (SEM)sulfonate
sulfonate (MRES);(B)
morphologies
(MRES); (B)
of polyvinyl
the alcohol
nanocapsules
polyvinyl alcohol (PVA);
(A)
(PVA); and
maleic
and (C)
(C) polycarboxylate (PC)).
rosinpolyoxypropylene-polyoxyethylene
polycarboxylate (PC)). ether sulfonate (MRES); (B) polyvinyl alcohol (PVA); and
(C) polycarboxylate (PC)).
Nanomaterials 2020, 10, 220 7 of 13

Nanomaterials 2020, 9, x FOR PEER REVIEW 7 of 13

3.2.2. Complex Surfactants
Surfactants
3.2.2. Complexare substances composed of hydrophilic and hydrophobic groups that can substantially
Surfactants
reduce the surface tension of a solution [47]. In recent years, surfactants have commonly been mixed
Surfactants are substances composed of hydrophilic and hydrophobic groups that can
to substantially
further improve reducetheirthe
performance by taking
surface tension of a advantage
solution [47].of their synergistic
In recent years, effects [48]. Correct
surfactants have
surfactant content plays an important role in stabilizing nanoparticles [49,50].
commonly been mixed to further improve their performance by taking advantage of their synergistic In the current study
weeffects
mixed[48].
the Correct
two selected
surfactant content plays an important role in stabilizing nanoparticles [49,50]. their
surfactants that demonstrated good emulsification effects and tested In
performance
the currentinstudy
different ratios (PVA:
we mixed the two = 1:1, 1:2,
PC selected 2:1, 1:3, and
surfactants that3:1). The MPS, diameters
demonstrated based on 90%
good emulsification
of effects
the cumulative
and testeddistribution profilein(D90),
their performance andratios
different PDI (PVA:
of the PCnanocapsules
= 1:1, 1:2, 2:1,obtained for the
1:3, and 3:1). Thedifferent
MPS,
ratios are presented in Table 1. All PVA:PC ratios yielded nanocapsules with
diameters based on 90% of the cumulative distribution profile (D90), and PDI of the nanocapsules uniform size within
theobtained
range 250–300 nm. Forratios
for the different each are
ratio, the PDIinwas
presented below
Table 1. All0.3, whichratios
PVA:PC implied goodnanocapsules
yielded nanocapsules’
dispersity [51]. size
with uniform The within
use of the
surfactant combinations
range 250–300 nm. For showed
each ratio,the potential
the PDI wasto yield0.3,
below nanocapsules
which impliedwith
good nanocapsules’ dispersity [51]. The use of
smaller particle size and better physical stability [52]. surfactant combinations showed the potential to yield
nanocapsules with smaller particle size and better physical stability [52].
Table 1. Effects of complex surfactants on the MPS, D90, and PDI of the nanocapsules.
Table 1. Effects of complex surfactants on the MPS, D90, and PDI of the nanocapsules.
PVA:PC MPS (d.nm) D90 (nm) PDI
PVA: PC MPS (d.nm) D90 (nm) PDI
1:1 287.4 ± 0.7 373.3 ± 26.3 0.051 ± 0.046
1:2
1:1 287.4 ± 0.7
265.3 ± 0.8
373.3 ± 26.3
330.7 ± 6.0
0.051 ± 0.046
0.034 ± 0.030
2:11:2 265.3±±0.2
279.7 0.8 330.7
359.7 ±± 6.0
14.5 0.034 ± 0.030
0.042 ± 0.002
1:32:1 279.7±±2.7
282.4 0.2 359.7
378.7±±14.5
8.0 0.042 ± 0.002
0.043 ± 0.018
3:11:3 282.4±±3.7
288.2 2.7 378.7
355.7 ±±8.0
8.7 0.043 ± 0.018
0.025 ± 0.017
3:1 288.2 ± 3.7 355.7 ± 8.7 0.025 ± 0.017
3.3. Morphology of Dual-Functionalized Pesticide Nanocapsules
3.3. Morphology of Dual-Functionalized Pesticide Nanocapsules
The nanocapsules prepared with different surfactant ratios were characterized by TEM, which
The nanocapsules prepared with different surfactant ratios were characterized by TEM, which
showed small spherical nanocapsules with regular morphology and distribution (Figure 4). Varying
showed small spherical nanocapsules with regular morphology and distribution (Figure 4). Varying
the surfactant ratio did not significantly affect the nanocapsules in terms of their particle size, dispersity,
the surfactant ratio did not significantly affect the nanocapsules in terms of their particle size,
or morphology. However, under the same conditions, the solid precipitate collected with a PVA:PC
dispersity, or morphology. However, under the same conditions, the solid precipitate collected with
ratio of 2:1 had a more uniform and smaller particle size. Thus, a PVA: PC surfactant ratio of 2:1 was
a PVA:PC ratio of 2:1 had a more uniform and smaller particle size. Thus, a PVA: PC surfactant ratio
selected to prepare
of 2:1 was selectedsamples forsamples
to prepare subsequent characterization.
for subsequent characterization.

Figure
Figure 4. 4.Transmission
Transmissionelectron
electron microscopy
microscopy (TEM)
(TEM) morphologies
morphologiesofofthe thenanocapsules
nanocapsules(A–E) and
(A–E) and
statistical dried particle size distributions of nanocapsules (a–e) based on the TEM images. (A–a,
statistical dried particle size distributions of nanocapsules (a–e) based on the TEM images. (A–a, B–b, B–b,
C–c,
C–c, D–d,
D–d, andand E–erepresent
E–e representPVA:
PVA:PCPC==1:1,
1:1, 1:2,
1:2, 2:1,
2:1, 1:3,
1:3, and
and 3:1,
3:1,respectively).
respectively).

3.4.3.4. Stability
Stability ofofDual-Functionalized
Dual-FunctionalizedPesticide
Pesticide Nanocapsules
Nanocapsules
Thestorage
The storagestability
stabilityofof pesticides
pesticides is
is an
an important
important indicator
indicatorininthetheevaluation
evaluationofofpesticide
pesticide
systems [53]. The MPS and PDI of the nanocapsules after storage in various conditions
systems [53]. The MPS and PDI of the nanocapsules after storage in various conditions were measured were
to measured
evaluate theto evaluate
physicalthe physical
stability of stability of thepesticide.
the complex complex pesticide. Afterof7 storage
After 7 days days of storage
at 0 C,atthe
◦ 0 °C,
MPS
the MPS decreased from 291.7 to 289.7 nm. After 14 days of storage
◦ at 25
◦ °C and
decreased from 291.7 to 289.7 nm. After 14 days of storage at 25 C and 54 C, the MPS decreased from54 °C, the MPS
decreased
291.7 from 291.7
nm to 284.5 nm tonm,
and 263.7 284.5 and 263.7 nm,
respectively respectively
(Figure 5). The(Figure 5). The MPS
MPS remained remained
relatively relatively
stable without
stable without significant changes throughout the storage process. In addition the PDIs of the
Nanomaterials 2020, 10, 220 8 of 13
Nanomaterials 2020, 9, x FOR PEER REVIEW 8 of 13

significant changes
nanocapsule throughout
suspensions werethe storage
below 0.3,process. In addition
indicating a uniformthesize
PDIs of the nanocapsule
distribution and goodsuspensions
dispersity
were below 0.3, indicating
of the nanocapsules [51]. a uniform size distribution and good dispersity of the nanocapsules [51].

Effects of
of temperature
temperatureon
onthe
theMPS
MPSand
andPDI
PDIofofthe
thenanocapslues.
nanocapslues.(A)
(A) ◦ ◦ C; and
Figure 5.
Figure 5. Effects 0 0°C;C;(B)
(B)25
25°C; and
(C) 54 ◦ C.
(C) 54 °C.
When stored at 0 ◦ C and 25 ◦ C, the MPS and PDI of the nanocapsule suspensions remained
When stored at 0 °C and 25 °C, the MPS and PDI of the nanocapsule suspensions remained
relatively stable, implying that the dual-functionalized pesticide nanocapsules had the desired physical
relatively stable, implying that the dual-functionalized pesticide nanocapsules had the desired
stability. However, storage at 54 ◦ C accelerated the process of the dual-functionalized pesticide
physical stability. However, storage at 54 °C accelerated the process of the dual-functionalized
nanocapsules approaching the glass transition temperature of PLA, which caused PLA to change from
pesticide nanocapsules approaching the glass transition temperature of PLA, which caused PLA to
a rigid state to a flexible state [54,55], resulting in a reduction in particle size. However, the pesticide
change from a rigid state to a flexible state [54,55], resulting in a reduction in particle size. However,
samples are not expected to experience such high temperatures under normal circumstances, so the
the pesticide samples are not expected to experience such high temperatures under normal
prepared nanocapsules are still regarded as having good storage stability.
circumstances, so the prepared nanocapsules are still regarded as having good storage stability.
3.5. Foliar Spread and Contact Angle Evaluation of Dual-Functionalized Pesticide Nanocapsules
3.5. Foliar Spread and Contact Angle Evaluation of Dual-Functionalized Pesticide Nanocapsules
The foliar spread and contact angle of the dual–functionalized pesticide nanocapsules on plant
The foliar spread and contact angle of the dual–functionalized pesticide nanocapsules on plant
leaves were characterized using an ESEM and a contact angle goniometer (Figure 6). The contact
leaves were characterized using an ESEM and a contact angle goniometer (Figure 6). The contact
angles of the blank control, dual-functionalized pesticide nanocapsules, and commercial VTSC, TSC,
angles of the blank control, dual-functionalized◦ pesticide nanocapsules, and commercial VTSC, TSC,
VAS, and VWP on cucumber leaves were 94.97 , 77.10◦ , 69.7◦ , 92.36◦ , 89.62◦ , and 77.96◦ , respectively.
VAS, and VWP on cucumber leaves were 94.97°, 77.10°, 69.7°, 92.36°, 89.62°, and 77.96°, respectively.
According to these data, the prepared nanocapsules showed no clear advantage compared with other
According to these data, the prepared nanocapsules showed no clear advantage compared with
groups. This observation is likely due to the fact that no additives such as sizing and spreading
other groups. This observation is likely due to the fact that no additives such as sizing and spreading
agent were added to the preparation of dual-functionalized pesticide nanocapsules [56]. However,
agent were added to the preparation of dual-functionalized pesticide nanocapsules [56]. However,
the ESEM images clearly show that the commercial formulations were spread on the leaf surface in
the ESEM images clearly show that the commercial formulations were spread on the leaf surface in
patches, whereas the dual-functionalized pesticide nanocapsules were scattered on the leaf surface
patches, whereas the dual-functionalized pesticide nanocapsules were scattered on the leaf surface
as uniform spheres. Compared with commercial formulations, the dual-functionalized pesticide
as uniform spheres. Compared with commercial formulations, the dual-functionalized pesticide
nanocapsules improved the foliar spreading behavior. As a result of the unique micro-nano structure,
nanocapsules improved the foliar spreading behavior. As a result of the unique micro-nano
the nanocapsules were able to embed into the leaf vein structure [9], which prevented the nanocapsules
structure, the nanocapsules were able to embed into the leaf vein structure [9], which prevented the
from collapsing and promoted pesticide absorption and transportation by the leaf. These qualities are,
nanocapsules from collapsing and promoted pesticide absorption and transportation by the leaf.
thereby, expected to effectively increase the utilization efficiency of the pesticides and consequently
These qualities are, thereby, expected to effectively increase the utilization efficiency of the
lead to a reduction of pesticide application dosage.
pesticides and consequently lead to a reduction of pesticide application dosage.
Nanomaterials 2020, 10, 220 9 of 13
Nanomaterials 2020, 9, x FOR PEER REVIEW 9 of 13
Nanomaterials 2020, 9, x FOR PEER REVIEW 9 of 13

Figure
Figure 6.6.Foliar
Figure6. Foliarspread
spreadand
spread andcontact
and contact
contactangle
angle ofdifferent
of
angle different
of pesticide
pesticide
different formulations
formulations
pesticide onon
formulations cucumber
cucumber
on leaves
leaves
cucumber (A) (A)
leaves
blank
blank
(A) control;
control;
blank (B)
control; dual-functionalized
(B)(B)
dual-functionalized pesticide
pesticide
dual-functionalized nanocapsules;
nanocapsules;
pesticide (C)
(C)
nanocapsules; VTSC;
VTSC;
(C) D:D:
VTSC; TSC;
TSC;
(D) E:(E)
E: VAS;
TSC; VAS; F:(F)
VWP).
F: VWP).
VAS; VWP.

3.6. Bioactivity
3.6.Bioactivity
3.6. ofofthe
Bioactivityof the
theDual-Functionalized Pesticide
Dual-Functionalized Pesticide
Dual-Functionalized Nanocapsules
PesticideNanocapsules
Nanocapsules
The
Thebioactivity
The bioactivity of
bioactivity of dual–functionalized
of dual–functionalized pesticide
dual–functionalized pesticide nanocapsules
pesticidenanocapsules
nanocapsuleswas was
was evaluated
evaluated
evaluated to to verify
verify
to the the
verify the
feasiblilityof
feasiblility
feasiblility ofapplying
of applying the
applying the new
thenewnewpesticide
pesticide
pesticide formulation.
formulation.
formulation. The
Theloading
The efficiencies
loading
loading forfor
efficiencies
efficiencies validamycin andand
for validamycin
validamycin
thifluzamide
and thifluzamide
thifluzamide inthe
in the nanocapsules
innanocapsules
the nanocapsules were
were 9.9%
wereand
9.9% 9.9%
and 15.8%,
andrespectively.
15.8%, 15.8%, The colony
respectively.
respectively. The growth
The
colony colony
growth of R. solani
growth
of of
R. solani
R.treated
solani with
treated the same
with concentration
the same of AIs
concentration is shown
of AIs inisFigure
shown 7. As
in
treated with the same concentration of AIs is shown in Figure 7. As shown in Table 2, the EC50 of shown
Figure in
7. Table
As 2,
shown the EC
in 50 of 2,
Table
VWP,
the
VWP, TSC, VWP,
ECTSC, thedual-functionalized
50 of the
dual-functionalized pesticide
pesticidenanocapsules,
TSC, the dual-functionalized pesticide and
nanocapsules, VTSC
nanocapsules,
and VTSC were
were227.5372,
and VTSC 0.0107,
227.5372, were 0.0082,
0.0107,227.5372,
0.0082,
and
0.0107, 0.0350
0.0082, μg/mL,
and respectively.
0.0350 µg/mL, These results
respectively. demonstrate
These results that the
demonstrate dual-functionalized
that the pesticide
dual-functionalized
and 0.0350 μg/mL, respectively. These results demonstrate that the dual-functionalized pesticide
nanocapsules
pesticide had a synergistic
nanocapsules effect compared
had a synergistic with VWPwith
effect compared andVWP TSC, andand TSC,
the toxicity theoftoxicity
the
nanocapsules had a synergistic effect compared with VWP and TSC, and theandtoxicity of the
ofnanocapsules
the nanocapsules was 4.2 times
wastimes greater
4.2 times than
greater that of VTSC. In addition, the SR values for the the
nanocapsules was 4.2 greater thanthan that that of VTSC.
of VTSC. In In addition,the
addition, theSR
SR values
values forfor the
dual-functionalized nanocapsuled and VTSC were 2.088 and 0.917, respectively. These results
dual-functionalized nanocapsuled and VTSC were 2.088 and 0.917,
dual-functionalized nanocapsuled and VTSC were 2.088 and 0.917, respectively. These results respectively. These results indicate
indicate that the two AIs in the dual-functionalized pesticide nanocapsules had a significant
that the two
indicate thatAIs thein two
the dual-functionalized
AIs in the alone pesticide nanocapsules
dual-functionalized pesticide had a significant synergistic
nanocapsules effect,
synergistic effect, whereas VTSC exhibited an additive effect between thehad two aAIs.significant
The
whereas
synergisticVTSC alone
effect, exhibited
whereas an
VTSC additive
alone effect between the two AIs. The nanocapsules were, therefore,
nanocapsules were, therefore, superior to exhibited
the commercial an additive
control in effect
termsbetween the twotoxicity
of both relative AIs. The
superior
nanocapsulesto thewere,
commercial
therefore, control in terms
superior to of commercial
the both relativecontrol
toxicityinandtermssynergistic
of both effect; which
relative is
toxicity
and synergistic effect; which is attributed to the unique micro-nano structure being able to facilitate
attributed
and
adsorption
to and
synergistic theeffect;
unique micro-nano
which
transportation
structure
is attributed
into fungaltocells thebeing
unique ableTherefore,
[57,58].
to facilitate
micro-nano adsorption
structure
the
andable
being
nanocapsules
transportation
to facilitate
substantially
into fungal
adsorption
improved and cells [57,58]. Therefore,
transportation
the antifungal into
activity the nanocapsules
of fungal substantially
cells [57,58].
the pesticides, whichTherefore, improved the antifungal
the nanocapsules
could potentially activity of
reduce the substantially
pesticide
the pesticides,
improved
application the which could potentially reduce the pesticide application rate.
antifungal activity of the pesticides, which could potentially reduce the pesticide
rate.
application rate.

Figure7.7.Colony
Figure Colonygrowth
growthofofRhizoctonia
Rhizoctoniasolani
solaniunder
undertreatment
treatment with
with different
different pesticide
pesticide preparations
preparations (A)
(A) blank control;
blank (B) VWP; (C) TSC;
(D) (D) dual-functionalized pesticide nanocapsules;andand(E)
(E)VTSC.
VTSC.
Figurecontrol; (B) VWP;
7. Colony growth(C)
ofTSC; dual-functionalized
Rhizoctonia pesticide
solani under treatment nanocapsules;
with different pesticide preparations
(A) blank control; (B) VWP; (C) TSC; (D) dual-functionalized pesticide nanocapsules; and (E) VTSC.
Table 2. Bioactivity of the dual-functionalized pesticide nanocapsules against Rhizoctonia solani.

Table 2. BioactivityToxicity Correlation pesticide nanocapsules against Rhizoctonia solani.

of the dual-functionalized
36-h EC50 (μg/mL) EC50 (μg/mL) Synergistic
Treatments Regression Coefficient
Toxicity Correlation (95% CI) (Theoretical) Ratio
Equation (R2) 36-h EC50 (μg/mL) EC50 (μg/mL) Synergistic
Treatments Regression Coefficient
Y = 0.8485x 227.5372
(95% CI) (Theoretical) Ratio
VWP Equation 0.9716
(R2) – –
+ 3.0276 (135.8436–339.1162)
TSC YY == 0.8485x
0.5736x 0.9802 227.5372
0.0107 – – –
VWP 0.9716 –
+ 3.0276 (135.8436–339.1162)
TSC Y = 0.5736x 0.9802 0.0107 – –
Nanomaterials 2020, 10, 220 10 of 13

Table 2. Bioactivity of the dual-functionalized pesticide nanocapsules against Rhizoctonia solani.

Correlation EC50
Toxicity Regression 36-h EC50 (µg/mL) Synergistic
Treatments Coefficient (µg/mL)
Equation (95% CI) Ratio
(R2 ) (Theoretical)
227.5372
VWP Y = 0.8485x + 3.0276 0.9716 – –
(135.8436–339.1162)
0.0107
TSC Y = 0.5736x + 6.1966 0.9802 – –
(0.0050–0.0222)
Dual–functionalized 0.0082
Y = 0.5736x + 6.1966 0.9974 0.01712 2.088
pesticide nanocapsules (0.0062–0.0110)
0.0350
VTSC Y = 0.5654x + 5.8325 0.9683 0.03210 0.917
(0.0176–0.0992)

4. Conclusions
In this study, nanocapsules loaded with two pesticides—thifluzamide and validamycin—were
successfully prepared based on a double emulsion method combined with high-pressure
homogenization technology. The effects of single surfactants and the mixing ratio of complex surfactants
on the dual-functionalized pesticide nanocapsules were investigated. The particle size, morphology,
loading efficiency, stability, foliar spread, and bioactivity of the nanocapsules were also analyzed.
The, nanocapsules showed better spreading performance on foliage compared with commercial
pesticide formulations. Compared with the commercial formulation, the dual-functionalized pesticide
nanocapsule delivery system demonstrated a synergistic effect, which significantly enhanced the
bioactivity against R. solani. This technology is expected to slow down the widespread pesticide
resistance problems by improving the effective utilization rate of pesticides while reducing the
pesticide input. In addition, this study provides new strategies for the further development of
new pesticide formulations. Future attempts will be addressed to the large–scale production of
the dual-functionalized pesticide nanocapsule delivery system, which could facilitate the industrial
application of this technology.

Author Contributions: J.C. and C.S. wrote the paper. A.W. and H.Z. provided assistance in experimental
operations and data analysis. H.C. and Y.W. provided the theoretical guidance. N.L., B.C. and F.G. searched
literature. Y.S. and C.W. provided assistance in the preparation process and the characterization of the nanocapsules.
X.Z. and Z.Z. provided assistance in the biological activity analysis. All others contributed with the references.
All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the National Key R&D Program of China (2016YFD0200500), the National
Natural Science Foundation of China (31701825), the Technology Program for Water Pollution Control and
Treatment (2017ZX07101003), the National Key R&D Program of China (2017YFD0201207, 2018YFD0200200) and
the Agricultural Science and Technology Innovation Program (CAAS–XTCX2016004).
Acknowledgments: The authors thank Institute of Plant Protection and Agricultural Product Quality and
Safety, Anhui Academy of Agricultural Sciences; Institute of Crop Sciences, Chinese Academy of Agricultural
Sciences; Department of Chemical Engineering, Tsinghua University; Technical Institute of Physics and Chemistry,
Chinese Academy of Sciences for the technical supports of the bioassay and characterization of the nanocapsule
delivery system.
Conflicts of Interest: The authors declare no conflict of interest.

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