pubs.acs.

org/acsagscitech Article

Eucalyptus Oil-Based Nanoemulsion: A Potent Green Nanowagon

for Controlled Delivery of Emamectin Benzoate
Amit Kumar, Rohini Kanwar, and Surinder K. Mehta*
Cite This: ACS Agric. Sci. Technol. 2021, 1, 76−88 Read Online

ACCESS Metrics & More Article Recommendations *

sı Supporting Information
Downloaded via UNIV OF CALIFORNIA SANTA BARBARA on May 16, 2021 at 17:00:38 (UTC).
See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

ABSTRACT: Formulating a biocompatible water-based pesticide and eliminating the use of toxic solvents encourage sustainable
agriculture practices. In this study, a eucalyptus oil-in-water nanoemulsion (NEm) was employed as a green and biocompatible
nanocarrier for the delivery of a water insoluble pesticide, emamectin benzoate (EB). NEm was prepared using low-energy emulsion
phase inversion (EPI) and high-energy probe sonication (PS) with the help of food grade emulsifiers Tween 20 and Span 80. The
effect of using a single or binary surfactant system and its effect on NEm droplet size and stability were investigated by measuring the
change in droplet diameter for three months. The morphology, shape, and size of the NEm were assessed using high-resolution
transmission electron microscopy, field emission scanning electron microscopy, and dynamic light scattering. Fourier transform
infrared spectroscopy was employed to confirm the successful encapsulation of EB in the eucalyptus oil (EuO) core. During the
investigation, the encapsulation efficiency of EPI NEm (91%) was higher than that of PS NEm (86%). The antiphotolysis and leaf
adhesion ability of the EuO NEm was found to be better than those of free EB and the commercial EB formulation. Both NEms
showed slow and sustained release compared to the commercial EB formulation, which shows a burst release. This implies EuO
NEm behaves as a better pesticide formulation than free EB and its commercial analogue. However, EPI NEm with a higher
encapsulation efficiency and a better release performance compared to those of PS NEm is considered a low-cost and easy-to-prepare
nanoformulation. Thus, EB-loaded EuO NEm can be considered as a formulation that is greener, free of organic solvents,
biocompatible, and easy to scale up for industrial application with promising sustainable agriculture development.
KEYWORDS: nanoemulsion, essential oil, sustained release, photolysis, sonication

1. INTRODUCTION generally comprised of toxic organic solvents and harmful

With an increase in the global population, the demand for food
has increased significantly. Agrochemicals (pesticides and
fertilizers) have been extensively used to facilitate crop growth,
crop yield, and abundant food supply. However, indiscriminate
overuse of chemical pesticides has resulted in environmental
degradation and posed a severe threat to animal and plant life.
Moreover, conventional chemical pesticide formulations have
several drawbacks such as pesticide resistance, photolytic
degradation, hydrolysis, leaching, air−water drift, soil absorp-
tion, and poor aqueous solubility.1 A major percentage of the
active ingredient (AI) is degraded or lost, and <1% of the AI
reaches the target site. This not only increases the required
amount of pesticide but also leads to the high exposure of toxic
pesticides to plants and animals. For instance, emamectin
benzoate (EB), a biopesticide, is one of the most popular
pesticides around the globe because of its low cost, low toxicity,
and high efficacy against lepidopteran and coleopteran pests. EB
has higher antipest activity and lower toxicity compared to those
of avermectin and other conventional pesticides.2,3 According to
a report, EB is 18−1500-fold more potent against some insects
when compared to other insecticides.4 However, its poor
aqueous solubility, burst release, and rapid degradation under
sunlight limit its application in agriculture. A study by Mushtaq
et al. shows a short life span of EB under natural sunlight in pond
water.5 Moreover, the commercial pesticide formulations are
emulsifiers.
To overcome these issues, there is a pressing need to develop a
green nanowagon system for the controlled release of EB,
thereby preventing its direct exposure to harsh environmental
conditions. Numerous distinct nanocarriers exist, such as natural
polymer carbon nanoparticle microspheres, nanoparticles,
nanocomposites, and nanodispersions that have been employed
to prevent photolysis of EB and to control its release.6−14
However, most of these methods utilized either a very complex
synthesis method or a toxic solvent. Formulating a low-cost,
easily scalable, and efficient EB formulation remains a challenge.
Among all of the available nanoassemblies, a nanoemulsion
(NEm) has garnered significant attention due to its cost-
effectiveness, smaller droplet size, large surface area, good
physical stability, optical clarity, and resistance against
flocculation, Ostwald ripening, creaming, and sedimentation.15
NEm is a kinetically stable colloidal dispersion of oil, water, and
surfactant with a droplet size of <500 nm.16 NEm increases the
solubility and bioavailability of hydrophobic pesticides to a great
Received: November 25, 2020
Revised: February 15, 2021
Accepted: February 19, 2021
Published: February 26, 2021

© 2021 American Chemical Society https://dx.doi.org/10.1021/acsagscitech.0c00061

76 ACS Agric. Sci. Technol. 2021, 1, 76−88
ACS Agricultural Science & Technology
Figure 1. Schematic representation of the preparation of EB-loaded EuO NEm and its sustained release behavior.
extent. However, several reported NEm-based agrochemical
formulations have some drawbacks, mainly because of the oil
phase being comprised of toxic organic solvents.
Thus, essential oil-based NEms that are green, eco-friendly,
and biocompatible have emerged as effective botanical
pesticides due to the presence of an active volatile component
in the essential oils and their potential larvicidal, antibacterial,
and antifungal properties. Recently, few essential oils like neem
oil, eucalyptus oil, clove oil, and lemon oil have been explored in
the food and agriculture industry.17−20 Among them, eucalyptus
oil (EuO), with its main volatile constituent being 1,8-cineol or
eucalyptol, has shown promising pesticidal properties against
Pseudomonas aeruginosa, Candida spp., Sitophilus oryzae, and
Tribolium castaneum.18,21
In this report, a green biocompatible eucalyptus oil-in-water
NEm has been formulated for the delivery of EB with enhanced
solubility, controlled release, and antiphotolysis activity
compared to those of the commercial analogue. EuO has been
used as an oil phase, Tween 20, and Span 80 as a food grade
emulsifier for solubilizing the EB. Two simple and easily scalable
methods, emulsion phase inversion (EPI) and probe sonication
(PS), have been employed for the synthesis of EB NEm and
compared to understand how different modes of preparation
affect the stability and control release of the formulation. The
developed NEm has been characterized for droplet size,
polydispersity index (PDI), storage stability, morphology, and
composition using dynamic light scattering (DLS), ultraviolet−
visible (UV−vis) spectroscopy, high-resolution transmission
electron microscopy (HR-TEM), field emission scanning
electron microscopy (FESEM), and Fourier transform infrared
spectroscopy (FTIR). The stability of the EuO-based NEm has
been evaluated by determining the Ostwald ripening and
coalescence droplet growth rate over time. The controlled
release of the EB from NEm has been systematically performed
under in vitro conditions. Most importantly, a comparison
between the developed EB-loaded NEm and commercial EB
formulation has been performed to showcase how well the
system behaves in the real field. Figure 1 represents the scheme
77
pubs.acs.org/acsagscitech Article
for the fabrication of EB-loaded NEm and the targeted
application.
2. MATERIALS AND METHODS
2.1. Materials. Polysorbate 20 (Tween 20, purity of >98%),
sorbitan monooleate (Span 80, purity of >98%), emamectin benzoate
(EB, purity of >98%), eucalyptus oil (cineole, 60%), and dialysis tubing
(12−14 kDa) were purchased from Sigma-Aldrich. Commercial EB
(5% EC) was obtained from the local supermarket. Methanol (HPLC
grade) and doubly distilled water were used for all of the experiments.
2.2. Methods. 2.2.1. Preparation of the Nanoemulsion.
2.2.1.1. Low-Energy Emulsion Phase Inversion (EPI). NEm was
prepared using the EPI method, a low-energy method in which the
aqueous phase is titrated into the oil phase at a constant temperature
with constant stirring. The oil phase was prepared by mixing surfactants
Tween 20 and Span 80 with the EuO at room temperature for 1 h at a
constant magnetic stirring rate of 500 rpm. Then the aqueous phase
comprising doubly distilled water was added dropwise at a rate of 2 mL/
min into the oil phase with a constant stirring rate of 500 rpm. This
method involves the catastrophic inversion of a water-in-oil nano-
emulsion into an oil-in-water nanoemulsion.
2.2.1.2. High-Energy Probe Sonication (PS). The oil phase, aqueous
phase, and water phase were mixed using a magnetic stirrer at 500 rpm
for 5 min at 25 °C to form a coarse emulsion. The prepared coarse
emulsion was then subjected to probe sonication for 5 min at an
amplitude of 50% with a 1 min on−off cycle to obtain the final oil-in-
water NEm. The composition of the oil phase and the aqueous phase
was kept the same as that of EPI.
2.2.2. Optimization. 2.2.2.1. Influence of the Surfactant:Oil Ratio
(SOR). The properties of NEm, including the size, PDI, and stability, are
directly linked to the amount of oil and surfactant used. The effect of an
increasing SOR was observed by varying the amount of Tween 20 and
fixing the amount of EuO in the oil phase at 100 mg. The SOR can be
calculated by using the formula
ws
SOR =
wo (1)
ww = 100 − (ws + wo) (2)
where ws, wo, and ww are the weight of the surfactant, oil, and water,
respectively. SORs of 0.5, 1, 2, and 3 were tested for NEm prepared
using both EPI and PS as shown in Table S1.
https://dx.doi.org/10.1021/acsagscitech.0c00061
ACS Agric. Sci. Technol. 2021, 1, 76−88
ACS Agricultural Science & Technology
2.2.2.2. Effect of HLB on NEm Structure. A change in the HLB value
using a different mixture of surfactant ratios can significantly decrease
the droplet size and increase the stability to the formulated NEm. To
investigate the effect of HLB on the NEm, Span 80 was used along with
Tween 20. The effective HLB value of a mixed surfactant system can be
calculated by using Griffin’s formula:
HLBmix = HLBT20 × M T20 + HLBS80 × MS80 (3)
where HLBmix is the effective HLB of the surfactant mixture, HLBT20 is
the HLB of Tween 20, HLBS80 is the HLB of Span 80, MT20 is the mass
fraction of Tween 20, and MS80 is the mass fraction of Span 80. HLB was
varied from 10 to 15, as this HLB region is suitable for the formation of
an oil-in-water NEm.
2.2.3. Preparation of the EB-Loaded Nanoemulsion. After
preliminary optimization, the most stable NEm was chosen for the
encapsulation of EB. EB (10 mg) was dissolved in the oil phase of the
selected NEm and stirred at 300 rpm and 40 °C to solubilize EB into the
EuO. After complete solubilization of EB into the oil phase, the
temperature was adjusted to 25 °C. The prepared oil phase was then
used to formulate NEm using preoptimized compositions in EPI and
PS.
2.2.4. Instability Mechanism Evaluation. NEm is generally prone to
instability upon undergoing Ostwald ripening, coalescence, floccu-
lation, and creaming. Ostwald ripening is generally the major
mechanism for NEm instability. In Ostwald ripening, larger droplets
grow at the expense of small droplets because of the molecular
dissolution of dispersed oil droplets into the continuous water phase.
Ostwald ripening can be calculated by using the LSW theory using the
equation22
dr 3 8CγV mD
w= = was then immersed in a glass jacket containing 50 mL of methanol and a
dt 9ρRT (4)
where w is the droplet growth rate, r is the radius of the droplet, T is the
temperature, Vm is the molar volume of the oil phase, R is the gas
constant, D is the diffusion coefficient of the oil phase dispersed in the
aqueous phase, γ is the interfacial tension, and C is the bulk phase
solubility of the dispersed phase in the continuous phase. When the
cube of the hydrodynamic radius (r) is plotted as a function of storage
time, the value of the slope of the linear fit will give the Droplet growth
rate (w).
Apart from Ostwald ripening, the collision of dispersed phase
droplets with each other can also be the instability mechanism for NEm,
which is termed coalescence. The increase in droplet growth due to
coalescence can be calculated using eq 5.
dr 4π
= dt
r3 3ν (5)
where r is the hydrodynamic radius of the dispersed phase droplet and ν
is the frequency with which droplets rupture per unit of the surface of
the film. Integrating the equation gives
1 1 8πν
= − t measured. EB-loaded NEm was placed in a beaker and weighed. The
r2 r 02 3 (6)
2.3. Characterization of NEm. 2.3.1. Particle Size and
Polydispersity Index. The particle size and PDI of the EB-loaded
NEm were determined using dynamic light scattering (Malvern
Zetasizer Nano S90 DLS, argon laser wavelength of 633 nm). All of
the measurements were performed in quintuplicate at a scattering angle
of 90° at 25 °C.
2.3.2. Morphology. The internal structure and particle size of the
formulated NEm were imaged using a high-resolution transmission
electron microscope (JEM-2100, JEOL Ltd.). NEm was diluted first
with distilled water and then negatively stained using 1%
phosphotungstic acid (pH 7) for 10 min. The sample was then placed
on a carbon-coated copper grid and air-dried before being subjected to
HR-TEM observation.
The morphology of the NEm samples was evaluated using FESEM
(Hitachi SU8010). The NEm sample was prepared by placing a drop of
78
pubs.acs.org/acsagscitech Article
NEm on a glass coverslip and allowing it to dry at 40 °C. After drying,
the sample was subjected to FESEM observation.
2.3.3. FTIR Analysis. The chemical composition of NEm was
determined qualitatively using an attenuated total reflection Fourier
transform infrared spectrophotometer (ATR-Nicolet IS10, Thermo
Scientific). The data were collected within the IR range of 4000−500
cm−1 without any dilution.
2.4. Stability Analysis. The storage stability of formulated NEm
systems was evaluated by storing the formulated samples at 25 and 4 °C.
The change in the hydrodynamic diameter and PDI was measured
using DLS. Moreover, the instability of NEm was evaluated by
measuring the increase in the droplet growth rate and polydispersity
due to Ostwald ripening and coalescence. This could lead to leakage of
the AI encapsulated in oil droplets over time. The content of
encapsulated EB inside the NEm core was also evaluated during
storage at 4, 25, and 54 °C. The concentration of EB on day 1 was
considered to be 100%, and for rest of days, it was evaluated with
respect to day 1.
2.5. Encapsulation Efficiency (EE) and Loading Capacity (LC).
Analytical grade EB was dissolved in a solution of methanol and water
[3:7 (v/v)] at concentrations of 1−100 ppm to obtain the calibration
curve. A UV−vis spectrophotometer (Thermo-Scientific Evolution
200) was used to measure the absorption of solutions over the
wavelength range of 200−600 nm. On the basis of the absorption
maximum at 245 nm, a linear standard curve of EB was obtained as
shown in Figure S1.
To determine the encapsulation efficiency of EB-loaded nano-
emulsions, the dialysis bag method was employed. EB-loaded NEm (1
mg/mL) was centrifuged for 15 min at a rate of 15000 rpm to remove
the free drug from the system. In addition, 2 mL of supernatant was
transferred into a dialysis bag clipped from both sides. The dialysis bag
water solution operating at 25 °C for 24 h. The amount of unentrapped
EB released over 24 h was estimated using a UV−vis spectropho-
tometer, and the EE and LC of the NEm were calculated as follows:23
E1 − E2
EE (%, w/w) = × 100
(7)
E1
E1 − E2
LC (%, w/w) = × 100
(8)
E3
where E1 and E2 are the actual amounts of loaded and unentrapped EB,
respectively, and E3 is the amount of EuO. The experiments were
performed in triplicate.
2.6. Investigation of the Adhesion Performance of NEm on a
Foliar Surface. Leaf adhesion of a pesticide formulation is one of the
major parameters used to measure the performance and function of the
formulation. The amount of formulation retained on leaves also
depends on the hydrophilic and hydrophobic nature of the plant leaves.
The amount of EB-loaded NEm liquid retained on the surface of the leaf
was investigated using the microweighing and dipping method. In brief,
fresh hydrophobic and hydrophilic leaves were cut down into small
square pieces and their surface area A (square centimeters) was
leaf pieces were dipped in the formulation for 30 s, removed, and hung
over a beaker until no liquid drops fell from the leaf surface. Then the
leaf was placed aside, and the beaker was weighed again. The amount of
liquid retained on the leaf, W (milligrams), was measured. Experiments
were performed in triplicate. Leaf retention was calculated using the
formula
W
leaf retention = (mg/cm 2)
A (9)
2.7. Release Performance of EB. The pesticide release perform-
ance of NEm was investigated using a dialysis bag method. Two
milliliters of EB-loaded NEm (1 mg/mL) was first transferred to the
dialysis bag, and then the bag was clipped at both ends. The bag was
then immersed in 50 mL of solvent [3:7 (v/v) water/methanol] in a
glass jacket. The jacket was kept at room temperature (25 °C). Two
milliliters of solvent was withdrawn from the jacket and replaced with 2
https://dx.doi.org/10.1021/acsagscitech.0c00061
ACS Agric. Sci. Technol. 2021, 1, 76−88
ACS Agricultural Science & Technology
Figure 2. Effect of an increasing surfactant concentration on the hydrodynamic diameter and PDI of NEm formulated using (A) EPI and (B) PS. Effect
of preparation method on the (C) PDI and (D) hydrodynamic diameter of nanoemulsions.
mL of fresh solvent after every predesigned time interval. The
withdrawn sample volume was then analyzed for EB content using a
UV−vis spectrophotometer at a wavelength of 245 nm. All of the
experiments were performed in triplicate, and the cumulative release
curve of EB-loaded NEm was plotted. The release performance of EB
NEm was compared with that of the commercial EB formulation. The
release kinetics of EB was analyzed by fitting the cumulative release
profile data to a suitable kinetic model.
2.8. Antiphotolytic Performance Evaluation. EB is highly
photosensitive and rapidly degrades under sunlight. The photostability
performance of the EB-loaded NEms (1 mg/mL) was evaluated by
irradiating the samples under a UV lamp from a distance of 10 cm. The
remaining amount of EB in the samples was analyzed using a UV−vis
spectrophotometer at predetermined time intervals. Similarly, the
photostability of technical EB was also measured by dissolving EB in
methanol (1 mg/mL). The remaining percentage of EB after certain
irradiation time intervals was calculated using the formula
ij A yzz
residual rate percent = jjj1 −
zz × 100
j A 0 z{
k
(10)
where A0 is the initial absorbance of the solution.
3. RESULTS AND DISCUSSION
The choice of surfactant to formulate a greener nanowagon for
pesticide delivery is an important factor for sustainable
agriculture. Tweens and Spans are non-ionic surfactants that
are widely used in pharmaceutical, cosmetic, and food-related
formulations owing to their nontoxic nature and excellent
emulsification and solubilization properties. On the basis of the
solubility of Tween 20 being higher than those of other Tweens,
it was chosen as a primary emulsifier whereas Span 80 was
employed as a secondary emulsifier to improve the stability of
the NEm. The NEm was formulated using two methods, namely,
EPI and PS. High-energy methods like PS are efficient for
producing stable formulations but difficult to scale up to
79
pubs.acs.org/acsagscitech Article
commercialize the product, whereas a low-energy method like
EPI requires only simple mixing of organic and aqueous phases
and if optimized well can be scaled up easily. Herein, we
compare these two methods for fabrication, optimization, and
delivery of EB using biocompatible excipients. As droplet size
plays a vital role in the encapsulation and release of pesticides
and the stability of the formulated system, efforts were made to
prepare NEm with a droplet size of <200 nm with better
stability. It is expected that the smaller droplet size can help to
improve the stability of the pesticide-loaded formulated system.
3.1. Optimization of the EuO Nanoemulsion. 3.1.1. Ef-
fect of an Increasing Oil:Surfactant Ratio (SOR) on the EuO
Nanoemulsion. Food grade emulsifier Tween 20 was used as a
surfactant because of its high HLB value (16.7), which helps in
the formation of a stable oil-in-water NEm. The effect of an
increasing concentration of a surfactant, i.e., Tween 20, on the
droplet size and PDI of NEm was investigated while keeping the
amount of the oil phase fixed at 100 mg (Figures 2A,B). With an
increase in the amount of Tween 20 from 50 to 200 mg in the oil
phase, a significant decrease in the mean hydrodynamic
diameter of the oil droplet was observed along with a decrease
in its PDI. This is due to the hydrophilic nature of Tween 20, as
it shows more affinity for water and decreases the oil droplet size
by decreasing the interfacial tension between the water and oil
phase.
When the amount of Tween 20 was kept at 50 mg, the
nanoemulsion formed was white and opaque. However, with an
increase in the oil:surfactant ratio from 1:0.5 to 1:1 and 1:2, the
NEm changed to a translucent state due to a decrease in droplet
size.24 A decrease in the size of NEm takes place because, with an
increase in concentration, more surfactant is available at the
droplet surface to cover it. A lower PDI value corresponds to the
monodispersity of the system. With a further increase in the
amount of Tween 20 to 300 mg, an increase in the droplet
https://dx.doi.org/10.1021/acsagscitech.0c00061
ACS Agric. Sci. Technol. 2021, 1, 76−88
ACS Agricultural Science & Technology pubs.acs.org/acsagscitech Article

Table 1. Influence of HLB on the Nanoemulsion

surfactant (mg)
sample no. sample EuO (mg) Tween 20 Span 80 Smix SORmix water (g) HLB
EPI Method
1 E2-A 100 175 25 200 2 9.8 15.1
2 E2-B 100 150 50 200 2 9.8 13.6
3 E2-C 100 125 75 200 2 9.8 12.05
4 E2-D 100 100 100 200 2 9.8 10.5
5 E3-K 100 275 25 300 3 9.7 15.67
6 E3-L 100 250 50 300 3 9.7 14.63
7 E3-M 100 225 75 300 3 9.7 13.6
8 E3-N 100 200 100 300 3 9.7 12.57
9 E3-O 100 175 125 300 3 9.7 11.53
10 E3-P 100 150 150 300 3 9.7 10.5
PS Method
1 S1-A 100 87.5 12.5 100 1 9.8 15.15
2 S1-B 100 75 25 100 1 9.8 13.6
3 S1-C 100 62.5 37.5 100 1 9.8 12.05
4 S1-D 100 50 50 100 1 9.8 10.5
5 S2-A 100 175 25 200 2 9.7 15.15
6 S2-B 100 150 50 200 2 9.7 13.6
7 S2-C 100 125 75 200 2 9.7 12.05
8 S2-D 100 100 100 200 2 9.7 10.5

diameter in the case of EPI-formulated NEm was observed while
the droplet size of PS-formulated NEm decreased. Likewise, Teo
et al.25 and Jafari et al.26 have observed an increase in droplet size
as the concentration of Tween 20 was increased beyond 2.5%.
This was attributed to the presence of surfactant micelles in the
aqueous phase when Tween 20 was used at higher
concentrations. These micelles are formed from the unadsorbed
Tween 20, which in turn results in the flocculation of droplets
and destabilization of NEm. However, in the case of PS
formulated with the oil droplets with more NEm (Figure 2B),
the droplet size of NEm is smaler, which means that the surface
area provides more space for surfactant monomer adsorption.
Consequently, there would not be free Tween 20 micelles to
increase the size of the oil droplets.
3.1.2. Effect of Preparation Method on NEm Size and
Stability (EPI vs PS). For the successful formation of an oil-in-
water NEm, two methods, EPI and PS, were used. Generally,
more surfactant required for EPI than for PS. In EPI, Tween 20
was solubilized in the oil phase and then homogenized under
constant stirring. The aqueous phase was titrated into the oil
mixture, which initially produced a water-in-oil NEm, which on
continuous addition of aqueous phase undergoes a catastrophic
phase inversion and results in the formation of an oil-in-water
NEm.27 Using the EPI method, a smallest mean droplet size of
214 nm and a PDI of 0.2 were obtained at an SOR of 1:3.
In PS, the oil phase and aqueous phase were mixed to form a
coarse emulsion, which was further subjected to probe
sonication (amplitude of 40% for 5 min). In PS, ultrasonic
cavitation breaks down the large oil droplet of the coarse
emulsion into smaller ones by producing microbubbles in the
solution.28 Bursting of these microbubbles breaks down the
large oil droplets in a narrow nanosize distribution. At SORs of
1:2 and 1:3, PS could produce <200 nm droplets.
Panels C and D of Figure 2 show the comparison of the
droplet size and PDI of NEm prepared by EPI and PS. Upon
comparison of the droplet size and PDI of NEm (at the same
concentrations) prepared by the two methods, PS-formulated
NEm exhibited a smaller droplet size as well as a smaller PDI for
80
NEm than those produced by EPI. This can be attributed to the
high energy used in the PS method. Moreover, the PS method
could produce very small nanodroplets even at a low surfactant
concentration. However, at a high surfactant concentration,
both methods could form NEm with a small droplet size. The
PDI remained <0.3, which indicates the monomodal distribu-
tion of droplets.
3.1.3. Storage Stability. All of the prepared NEm systems
were subjected to stability tests by being stored at 25 and 4 °C
for a month. DLS is generally regarded as the best method for
testing the stability of NEm using the droplet size and PDI of the
NEm. Figure S2 depicts a comparison between the droplet size
and PDI of NEm after storage for 30 days at 25 and 4 °C,
respectively. DLS confirmed a significant increase in the droplet
size and PDI of NEm within 30 days and the destabilization of
NEm. Phase separation occurred at a 2:1 SOR within only 14
days, which may be attributed to the smaller amount of
surfactant, which fails to overcome the interfacial tension
between the oil phase and aqueous phase. As a result, droplet
aggregation occurs, which finally leads to creaming and visible
phase separation.24 A significant increase in the droplet size and
PDI was observed in the NEm prepared by EPI. Even the NEm
formulated by PS was destabilized, but the increase in droplet
size was found to be smaller as compared to that of the EPI-
produced NEm. This increase in droplet diameter and PDI may
be attributed to Ostwald ripening, flocculation, and creaming.
With an increase in the SOR (1:1, 1:2, and 1:3), more
surfactants became available and stopped creaming. Because of
the small size of these NEms, the Brownian motion of smaller
droplets becomes comparable to gravitational forces. A larger
amount of surfactant in the NEm overcomes the gravitational
separation that leads to creaming, but some other factors still
lead to the instability of the formulated NEm. Ostwald ripening
and coalescence are common instability mechanisms that cause
an increase in the droplet diameter, which ultimately leads to
flocculation or phase separation. Due to the considerable
increase in the droplet diameter of NEm with time, the
formulations become nonhomogeneous after 1 month, and the
https://dx.doi.org/10.1021/acsagscitech.0c00061
ACS Agric. Sci. Technol. 2021, 1, 76−88
ACS Agricultural Science & Technology
Figure 3. Effect of HLB on PDI and droplet size of nanoemulsions formulated using SORmix values of (A) 1:3 and (B) 1:2 by EPI and (C) 1:1 and (D)
1:2 by PS.
increase in the PDI value suggests that a multiple-particle size
population is present.
3.2. Effect of Changing the HLB Using a Binary
Surfactant System on the Stability of NEm. Because a
single-surfactant system was not able to stabilize the NEm for a
long period, lipophilic surfactant Span 80 was used in
combination with Tween 20. Moreover, a higher concentration
of surfactant is regarded as toxic (and costly). To obtain the best
surfactant mixture ratio that can stabilize the NEm, the
oil:surfactant mixture (SORmix) ratio was optimized by
evaluating the change in droplet diameter and PDI. Table 1
represents the composition of all surfactant mixture ratios.
3.2.1. Effect of the Change in HLB on EPI-Formulated NEm.
Because EPI is a low-energy method and generally requires a
large amount of surfactant, only SORmix values of 1:2 and 1:3
were selected for EPI. Panels A and B of Figure 3 depict the
change in the droplet diameter and PDI values of NEm with
respect to the change in the HLB values of the mixed surfactant
system. The NEm system with HLBs of 10−12 showed the
smallest droplet diameter. As compared to those of a single-
surfactant NEm, the droplet diameter and interfacial tension of
NEm decreased upon addition of Span 80. For a 1:2 SORmix, the
NEm corresponding to HLB values of 12.05 and 10.5 showed
the smallest droplet diameter, that is, 47.1 and 58.65 nm,
respectively. For a 1:3 SOR, NEm corresponding to HLB values
of 11.5 and 10.5 showed the smallest droplet diameters, that is,
32.67 and 41.94 nm, respectively.
3.2.2. Effect of the Change in HLB on PS-Formulated NEm.
PS is a high-energy method and can produce small droplets even
at low surfactant concentrations. Moreover, a low surfactant
concentration is always preferred for a biocompatible nano-
81
pubs.acs.org/acsagscitech Article
carrier. As a result, SORmix values of 1:1 and 1:2 were selected to
evaluate the influence of the change in HLB as shown in panels
C and D of Figure 3. Using the PS method, all of the NEm with
an SOR of 1:2 showed a droplet diameter of <100 nm in the
HLB range of 10−15. For an SOR of 1:2, the NEm
corresponding to HLB values of 15.1 and 13.6 showed the
smallest droplet sizes of 62.04 and 52.63 nm, respectively. NEm
with an SOR of 1:1 showed a droplet diameter of <160 nm. For
an SOR of 1:1, NEm corresponding to HLB values of 12.05 and
10.5 exhibited the smallest droplet sizes of 142.7 and 155 nm,
respectively. Further investigation of NEm stability was
performed on the basis of storage stability.
3.2.3. Stability of Binary Surfactant-Stabilized NEm. The
storage stability of pesticide formulations is an important factor
for their commercial use. The stability studies of binary
surfactant-stabilized NEm were performed by storing them at
25 °C for three months, and the change in droplet size and PDI
was measured using DLS. Generally, Ostwald ripening and
coalescence are the main instability mechanisms by which NEm
becomes destabilized. The droplet growth rate was monitored,
and the instability mechanism was evaluated for all NEm
systems. The formulations showing phase separation within one
month of storage time are not evaluated further.
3.2.3.1. Ostwald Ripening of EPI-Formulated NEm. Panels
A and B of Figure 4 depict the change in the droplet diameter of
EPI NEm systems and their Ostwald ripening rate for three
months, respectively. The E2-A, E2-B, and E3-K systems
showed excellent stability and the smallest change in droplet size
with the passage of time compared to those of other systems.
With the linear fitting of the data for the first 30 days, the droplet
https://dx.doi.org/10.1021/acsagscitech.0c00061
ACS Agric. Sci. Technol. 2021, 1, 76−88
ACS Agricultural Science & Technology
Figure 4. (A) Change in the droplet diameter of EPI nanoemulsions and (B) evaluation of their Ostwald ripening rates. (C) Change in the droplet
diameter of PS nanoemulsions and (D) evaluation of their Ostwald ripening rates.
growth rate was calculated by determining the value of the slope.
All of the obtained parameters are summarized in Table S2.
The droplet growth rate was found to be very slow in the case
of the E2-A, E2-B, and E3-K systems. The lowest droplet growth
rate for E3-K NEm might be attributed to the high surfactant
concentration and very small particle size of the NEm. In
general, it is expected that the droplet growth rate of E3-K
should increase as the surfactant concentration was high in E3-K
NEm as compared to those in E2-A and E2-B NEm,29,30 but the
Ostwald ripening rate decreases. Liu and co-workers31 have also
achieved a similar decrease in the Ostwald ripening rate with an
increase in the amount of surfactant, while they were
formulating a paraffin oil-in-water NEm using Tween 20 and
Span 80 by EPI. The decrease in the Ostwald ripening rate was
thought to be caused by the presence of free surfactant micelles
in the aqueous phase. Actually, after the droplet surface area had
been covered, the extra surfactant present in the aqueous phase
forms micelles, which prevents the oil from diffusing into the
water phase, which is a prerequisite condition for Ostwald
ripening in an oil-in-water NEm, and ultimately decreases the
droplet growth rate.
3.2.3.2. Ostwald Ripening of PS-Formulated NEm. The
increases in droplet size in the S2-A and S2-B systems were very
small. A small increase in size was noticed for the first 30 days,
and then the size became almost constant (Figure 4C). When
the cube of the radius (r3) was plotted against storage time, a
linear relationship with a correlation of >0.95 was obtained for
both systems. This suggests that the increase in the droplet size
of NEm was due to the Ostwald ripening mechanism. In
contrast, the droplet size of S1-A and S1-B systems showed an
82
pubs.acs.org/acsagscitech Article
irregular trend, first a decrease after storage for one week and
then a significant increase in size (Figure 4C). As the increase
was not linear, the instability mechanism cannot be to Ostwald
ripening. The increase in the droplet diameter of the S1-A and
S2-A systems was exponential and suggests that coalescence by
flocculation is the mechanism behind the instability. This might
be due to the presence of a smaller amount of surfactant that was
unable to cover the surface of oil drops or stabilize against
coalescence or phase separation. Table S4 depicts the various
parameters obtained from the evaluation of the Ostwald
ripening of the NEm.
Comparing the two methods, we can conclude that the
systems formulated using PS were more stable against phase
separation than those formulated using EPI. Moreover, the NEm
obtained using PS was smaller than the NEm obtained using
EPI. This is because of the high energy that was used in PS to
break the droplets into the nano range. From the evaluation of
Ostwald ripening, all of the NEm systems, including E2-A, E2-B,
E3-K, S2-A, and S2-B, showed very good stability against
ripening as the droplet growth rate was very small. Even the
droplet growth rate of PS-formulated NEm was small compared
to that of EPI-formulated NEm.
In addition to Ostwald ripening, coalescence is one of the
common instability mechanisms in NEm. To evaluate the
coalescence rate, 1/r2 was plotted against storage time (Figure
S3), which showed a linear decrease in 1/r2 with storage time
after 21 days for EPI and PS NEm.32 After the stability
evaluation of NEm based on Ostwald ripening and coalescence,
it was found that the increase in the droplet size of E2-A, S2-A,
E2-A, E2-B, and E3-K NEm was due to Ostwald ripening for the
https://dx.doi.org/10.1021/acsagscitech.0c00061
ACS Agric. Sci. Technol. 2021, 1, 76−88
ACS Agricultural Science & Technology pubs.acs.org/acsagscitech Article

Figure 5. Change in EB content over a storage period of 20 days at different temperatures: (A) S2-A NEm and (B) E2-A NEm.

Figure 6. (A) DLS particle size distribution of (I) E2-A and (II) S2-A NEm. (B) TEM micrograph of (a) EB-loaded E2-A NEm and (b) S2-A NEm.
(C) FTIR spectra of EB, S2-A NEm, and E2-A NEm. (D) Encapsulation efficiency and loading capacity of formulated NEms. (E) Affinities on
hydrophobic and hydrophilic leaves.

first few weeks followed by coalescence in later stages. Koroleva
et al.33 also obtained similar results for NEm stabilized using
non-ionic surfactants. Consequently, we may conclude that
Ostwald ripening is the major instability mechanism for the
83
increase in droplet size in S2-A, S2-B, E2-A, E2-B, and E3-K
NEms. Due to lower droplet growth rate, optical clarity, and
long-term stability, the S2-A and E2-A systems were chosen for
further encapsulation of the pesticide. The E3-K NEm system
https://dx.doi.org/10.1021/acsagscitech.0c00061
ACS Agric. Sci. Technol. 2021, 1, 76−88
ACS Agricultural Science & Technology
Figure 7. (A) Antiphotolytic performance of the formulations. (B) Pesticide release performance of commercial EB, S2-A, and E2-A. Fitting of release
data to (C) first-order, (D) Hixson−Crowell, (E) Higuchi, and (F) Korsmeyer−Peppas release models.
was not evaluated further for pesticide loading as the surfactant
content was higher in this system than in other stable systems. A
nanocarrier system with a lower surfactant content without
compromising stability is considered the best.
3.2.3.3. Influence of Storage Conditions on the Entrapped
EB Content. The EB content in E2-A and S2-A NEms at
different storage temperatures, that is, 4, 25, and 54 °C, for a
period of 20 days is depicted in Figure 5. A smaller change in EB
content was observed for the NEm formulations when they were
stored at 4 and 25 °C in comparison to 54 °C. After storage for
20 days, both S2-A and E2-A NEms at 4 and 25 °C were able to
retain >75% of EB while the amount decreased to 65.8% and
67.87% for S2-A and E2-A, respectively, at 54 °C. This can be
attributed to an increase in the ripening rate with an increase in
temperature, which changes the solubility of dispersed phase
and its diffusivity.34 Figure S4 depicts the change in size of EB-
loaded NEm over the different storage periods.
84
pubs.acs.org/acsagscitech Article
3.3. Characterization of EB-Loaded NEm. 3.3.1. Micro-
scopic Analysis. The surface morphology and droplet size
distribution of EB-loaded NEm were determined using DLS,
FESEM, and HR-TEM as depicted in panels A and B of Figure 6.
The particle size of E2-A (100 ± 3 nm) NEm was found to be
larger than that of S2-A (20−50 nm) NEm (Table S3). The PDI
of both NEm systems was <0.3, which confirms the
monodispersity of NEm systems. The HR-TEM micrographs
confirmed that the NEm consists of spherical particles. The
particle size of NEm was analyzed using ImageJ 1.46R. FESEM
reveals the spherical surface morphology of NEm (Figure S5).
3.3.2. FTIR Analysis. The FTIR spectra of EB and EB-loaded
NEm are shown and compared in Figure 6C. For technical EB,
the peaks at 2965 and 2931 cm−1 correspond to the C−H
vibration of the aromatic ring (benzoate fraction or conjugated
olefins) and the peaks at 1635, 1596, and 1558 cm−1 (CC
stretching vibrations of the aromatic ring or conjugated olefin),
https://dx.doi.org/10.1021/acsagscitech.0c00061
ACS Agric. Sci. Technol. 2021, 1, 76−88
ACS Agricultural Science & Technology
Figure 8. Pesticide release mechanisms at different stages.
1455 and 1376 cm−1 (C−H skeleton vibration), 1166, 1111, and
1054 cm−1 (C−C stretching vibration, OH flexion, and C−O−
C flexion), 1000 cm−1 (C−H flexion of trans CC bonding),
and 940−528 cm−1 (C−H flexion outside the plane of an
aromatic ring or CC cis bond) are assigned to EB. The
characteristic peaks of EB are appearing in the EB-loaded NEm
formulations, indicating the successful loading of EB in the
NEm.
3.3.3. EE and LC (percent). A nanowagon should be able to
carry and deliver a significant amount of an AI. In this study,
both EuO NEm formulations showed very high encapsulation
and loading capacities. The EE of E2-A (91%) was found to be
higher than that of S2-A (84%) (Figure 5D). Moreover, the
loading capacity of E2-A was also larger than that of S2-A NEm.
This may be attributed to the smaller particle size of S2-A NEm.
3.4. Leaf Affinity of EB-Loaded NEm. After a pesticide
formulation had been sprayed, the formulation needs to adhere
to the leaf surface to be effective. Basically, the higher affinity of
the formulation for the leaf surface and the reduced loss of
85
pubs.acs.org/acsagscitech Article
pesticide in water from the leaf surface are thus desired. In this
study, the affinity of EB-loaded NEm was evaluated against
hydrophilic Cucurbita moschata (pumpkin) leaves and hydro-
phobic Epipremnum aureum (Money plant) leaves. The affinity
of the liquid NEm formulation for leaves was evaluated by
calculating the amount of residual EB-loaded NEm liquid on the
leaf surface. The results are depicted in comparison with the
value of the commercial EB formulation in Figure 6E. Upon
comparison of the leaf retention affinity, the retention of the
liquid EB formulation by EPI (E2-A)- and PS (S2-A)-
formulated NEm was almost the same. Upon comparison of
the retention affinity of the commercial EB formulation, the
affinity was increased 1.4−1.5-fold in the case of EB-loaded
NEm on both hydrophilic and hydrophobic leaves. These results
depict that EB-loaded NEms have a stronger affinity for both
hydrophilic and hydrophobic leaves.
3.5. Antiphotolytic Performance of EB-Loaded NEm.
The antiphotolytic performance of EB-loaded NEm was
assessed by evaluating the residual percentage of EB in
https://dx.doi.org/10.1021/acsagscitech.0c00061
ACS Agric. Sci. Technol. 2021, 1, 76−88
ACS Agricultural Science & Technology
formulations after UV irradiation. As shown in Figure 7A, just
<19% of EB remained after UV light exposure for 6 h (mercury
lamp, 125 W MV, height of 10 cm) in the case of technical EB,
whereas for NEm formulations, 70% and 67.47% of EB were left
behind for E2-A and S2-A, respectively, after UV light irradiation
for 7 h. The slow degradation rate of EB in the case of NEm can
be attributed to the EuO core and mixed surfactant adsorbed at
the oil droplets that thwart EB from being rapidly degraded.
These results confirm that the EuO NEm is an excellent
nanowagon system for enhancing the antiphotolytic perform-
ance of EB and can be of great value for practical application in
agricultural fields.
3.6. Release Profile. The release performance of EB-loaded
NEm is an important factor for its practical application in
agricultural fields. The release performance of EB-loaded NEm
was compared with that of commercial EB. The dialysis method
was used to evaluate the pesticide release performance. Because
EB is insoluble in water, a 3:7 (v/v) methanol/water mixture
was used as a solvent system. The pesticide release study was
performed at 25 °C. The release behavior of the NEm systems is
presented in Figure 7B. Commercial EB showed burst release,
and 66% of the EB pesticide was released from the formulation
within 1 h. In just 24 h, >93% of the EB was released from the
commercial EB formulation. However, compared to commercial
EB, EuO NEm showed slow and sustained release behavior for
120 h. Table S4 compares the amount of EB released by the
commercial EB formulation and EuO NEms, namely, E2-A and
S2-A. We found that the EuO NEm systems are more efficient
for encapsulation and sustained release of EB than the
commercially available EB.
To investigate the kinetics and mechanism involved in the
release of EB from NEm, the experimental release data of both
NEm systems, E2-A and S2-A, were analyzed by first-order,
Higuchi, Hixson−Korwell, and Korsmeyer−Peppas (KP)
kinetic release models (panels C−F, respectively, of Figure 6).
Values of the parameters obtained from fitting models are listed
in Table S5. The KP model best fits the pesticide release data
with an R2 of >0.99. Hence, the KP model empirical equation35
was used to evaluate the release mechansim:
Ft
F= = k pt n
Fα (11)
where Ft/Fα denotes the percentage of EB released at time t, n is
the diffusional exponent, and kp is the Korsemeyer−Peppas
constant. An n value of <0.43 (Table S5) was obtained for the
S2-A and E2-A formulations, which implies that the pesticide
transport mechanism for both NEm formulations involves
Fickian diffusion. The value of kp was greater for S2-A (22.516)
than for E2-A (14.84); i.e., the release rate is faster in the S2-A
NEm formulation than in E2-A NEm.
Furthermore, to investigate the release mechanism behavior
at different stages, the amount of EB released at different stages
was evaluated using a nonlinear least-squares method as
presented in Figure 8. Upon comparison of the initial burst
release for both systems, the values of diffusional exponent n for
E2-A and S2-A NEm in stage I were 0.36 and 0.27, respectively,
which suggests that EB was released via Fickian diffusion. The
burst release might be attributed to the direct exposure of the
formulation to the solvent medium.36 Basically, EB adsorbed at
the surface of oil droplets or surfactants immediately diffuses
into the release medium, resulting in a burst release. Moreover,
upon comparison of the EB release in stage II, both NEm
86
pubs.acs.org/acsagscitech Article
formulations showed slow and sustained release. The EB
released during the sustained release stage is present at the core
of the oil droplet. EPI-formulated E2-A NEm performed better
as a slow release formulation than PS-formulated S2-A.
In summary, a green nanowagon for delivery of EB was
formulated using eucalyptus oil as the oil phase and food grade
emulsifiers, Tween 20 and Span 80, as mixed surfactants. An
oil:surfactant ratio of 1:2 with a Span 80:Tween 20 ratio of 0.142
was found to be most suitable for the formation of a stable
nanoemulsion. The spherical shape of NEm was confirmed
using HR-TEM. An extremely small value of Ostwald ripening
and coalescence rate affirmed the good stability of the
formulated nanoemulsions. When compared to the commercial
EB formulation, the EuO NEm showed slow and sustained
release over time. Both EB-loaded NEms have shown more
affinity for the foliar surface compared to that of the commercial
EB formulation. The antiphotocatalytic performance of both
NEms was excellent compared to that of free EB. EPI was found
to be more suitable than PS because of its smaller energy input,
easy scale-up, and greater eco-friendliness. The formulation
synthesized using EPI showed more encapsulation as well as
more sustained release compared to the PS method. Thus, the
obtained EuO NEm can act as a biocompatible, greener, and
control delivery system for the delivery of a hydrophobic
pesticide, EB. In addition, the real time field application of the
formulated nanoemulsion, i.e., the effect of EuO on the
insecticidal activity of EB, will be worth instigating in our future
study.
■ ASSOCIATED CONTENT
* Supporting Information
sı
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsagscitech.0c00061.
Effect of an increasing SOR on NEm (Table S1),
parameters obtained from the evaluation of Ostwald
ripening of NEm (Table S2), comparison of DLS and
TEM sizes of nanoemulsions (Table S3), cumulative
release comparison at different time intervals (Table S4),
results obtained from different drug release models
(Table S5), standard calibration curve of EB (Figure
S1), change in the droplet diameter and PDI of NEm at
different storage temperatures (Figure S2), evaluation of
the coalescene rate of NEm (Figure S3), storage stability
of EB-loaded NEm over two months (Figure S4), and
FESEM images of NEm (Figure S5) (PDF)
■
AUTHOR INFORMATION
Corresponding Author
Surinder K. Mehta − Department of Chemistry and Centre of
Advanced Studies in Chemistry, Panjab University,
Chandigarh 160014, India; orcid.org/0000-0002-1623-
7745; Email: skmehta@pu.ac.in
Authors
Amit Kumar − Department of Chemistry and Centre of
Advanced Studies in Chemistry, Panjab University,
Chandigarh 160014, India; orcid.org/0000-0002-3671-
5601
Rohini Kanwar − Department of Chemistry and Centre of
Advanced Studies in Chemistry, Panjab University,
Chandigarh 160014, India
Complete contact information is available at:
https://dx.doi.org/10.1021/acsagscitech.0c00061
ACS Agric. Sci. Technol. 2021, 1, 76−88
ACS Agricultural Science & Technology
https://pubs.acs.org/10.1021/acsagscitech.0c00061
Funding
A.K. acknowledges DST, New Delhi, for an INSPIRE Junior
Research Fellowship (JRF). S.K.M. acknowledges DST-PURSE
II.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
The Sophisticated Analytical Instrumentation Facility (SAIF) of
Panjab University is thankfully acknowledged.
■
REFERENCES
(1) Nuruzzaman, M.; Rahman, M. M.; Liu, Y.; Naidu, R. Nano-
encapsulation, Nano-Guard for Pesticides: A New Window for Safe
Application. J. Agric. Food Chem. 2016, 64 (7), 1447−1483.
(2) Khan, M. M.; Nawaz, M.; Hua, H.; Cai, W.; Zhao, J. Lethal and
Sublethal Effects of Emamectin Benzoate on the Rove Beetle, Paederus
Fuscipes, a Non-Target Predator of Rice Brown Planthopper,
Nilaparvata Lugens. Ecotoxicol. Environ. Saf. 2018, 165, 19−24.
(3) Lumaret, J.-P.; Errouissi, F.; Floate, K.; Rombke, J.; Wardhaugh, K.
A Review on the Toxicity and Non-Target Effects of Macrocyclic
Lactones in Terrestrial and Aquatic Environments. Curr. Pharm.
Biotechnol. 2012, 13 (6), 1004−1060.
(4) Jansson, R. K.; Brown, R.; Cartwright, B.; Cox, D.; Dunbar, D. M.;
Dybas, R. A.; Eckel, C.; Lasota, J. A.; Mookerjee, P. K.; Norton, J. A.;
Peterson, R. F.; Starner, V. R.; White, S. Emamectin Benzoate : A Novel
Avermectin Derivative for Control of Lepidopterous Pests. Proceedings:
The Management of Diamondback Moth and Other Crucifer Pests 1997,
171−177.
(5) Mushtaq, M.; Chukwudebe, A. C.; Wrzesinski, C.; Allen, L. R. S.;
Luffer-Atlas, D.; Arison, B. H. Photodegradation of Emamectin
Benzoate in Aqueous Solutions. J. Agric. Food Chem. 1998, 46 (3),
1181−1191.
(6) Huang, G.; Qian, G.; Yan, Y.; Xu, D.; Xu, C.; Fu, L.; Lin, B. A Super
Long-Acting and Anti-Photolysis Pesticide Release Platform through
Self-Assembled Natural Polymer-Based Polyelectrolyte. React. Funct.
Polym. 2020, 146, 104429.
(7) Zhang, S. F.; Chen, P. H.; Zhang, F.; Yang, Y. F.; Liu, D. K.; Wu, G.
Preparation and Physicochemical Characteristics of Polylactide Micro-
spheres of Emamectin Benzoate by Modified Solvent Evaporation/
Extraction Method. J. Agric. Food Chem. 2013, 61 (50), 12219−12225.
(8) Song, S.; Wang, Y.; Xie, J.; Sun, B.; Zhou, N.; Shen, H.; Shen, J.
Carboxymethyl Chitosan Modified Carbon Nanoparticle for Con-
trolled Emamectin Benzoate Delivery: Improved Solubility, PH-
Responsive Release, and Sustainable Pest Control. ACS Appl. Mater.
Interfaces 2019, 11 (37), 34258−34267.
(9) Chen, C.; Zhang, G.; Dai, Z.; Xiang, Y.; Liu, B.; Bian, P.; Zheng, K.;
Wu, Z.; Cai, D. Fabrication of Light-Responsively Controlled-Release
Herbicide Using a Nanocomposite. Chem. Eng. J. 2018, 349 (March),
101−110.
(10) Xu, C.; Cao, L.; Zhao, P.; Zhou, Z.; Cao, C.; Li, F.; Huang, Q.
Emulsion-Based Synchronous Pesticide Encapsulation and Surface
Modification of Mesoporous Silica Nanoparticles with Carboxymethyl
Chitosan for Controlled Azoxystrobin Release. Chem. Eng. J. 2018, 348
(April), 244−254.
(11) Gao, Y.; Xiao, Y.; Mao, K.; Qin, X.; Zhang, Y.; Li, D.; Zhang, Y.;
Li, J.; Wan, H.; He, S. Thermoresponsive Polymer-Encapsulated
Hollow Mesoporous Silica Nanoparticles and Their Application in
Insecticide Delivery. Chem. Eng. J. 2020, 383, 123169.
(12) Gao, Y.; Zhang, Y.; He, S.; Xiao, Y.; Qin, X.; Zhang, Y.; Li, D.; Ma,
H.; You, H.; Li, J. Fabrication of a Hollow Mesoporous Silica Hybrid to
Improve the Targeting of a Pesticide. Chem. Eng. J. 2019, 364, 361−
369.
(13) Shoaib, A.; Waqas, M.; Elabasy, A.; Cheng, X.; Zhang, Q.; Shi, Z.
Preparation and Characterization of Emamectin Benzoate Nano-
87
pubs.acs.org/acsagscitech Article
formulations Based on Colloidal Delivery Systems and Use in
Controlling: Plutella Xylostella (L.) (Lepidoptera: Plutellidae). RSC
Adv. 2018, 8 (28), 15687−15697.
(14) Cui, J. G.; Mo, D. M.; Jiang, Y.; Gan, C. F.; Li, W. G.; Wu, A.; Li,
X. Y.; Xiao, J. A.; Hu, Q.; Yuan, H. Y.; Lu, R.; Huang, Y. M. Fabrication,
Characterization, and Insecticidal Activity Evaluation of Emamectin
Benzoate-Sodium Lignosulfonate Nanoformulation with PH-Respon-
sivity. Ind. Eng. Chem. Res. 2019, 58 (43), 19741−19751.
(15) Montes de Oca-Á valos, J. M.; Candal, R. J.; Herrera, M. L.
Nanoemulsions: Stability and Physical Properties. Curr. Opin. Food Sci.
2017, 16, 1−6.
(16) McClements, D. J. Nanoemulsions versus Microemulsions:
Terminology, Differences, and Similarities. Soft Matter 2012, 8 (6),
1719−1729.
(17) Campos, E. V. R.; de Oliveira, J. L.; Pascoli, M.; de Lima, R.;
Fraceto, L. F. Neem Oil and Crop Protection: From Now to the Future.
Front. Plant Sci. 2016, 7, 1−8.
(18) Adak, T.; Barik, N.; Patil, N. B.; Govindharaj, G. P. P.; Gadratagi,
B. G.; Annamalai, M.; Mukherjee, A. K.; Rath, P. C. Nanoemulsion of
Eucalyptus Oil: An Alternative to Synthetic Pesticides against Two
Major Storage Insects (Sitophilus Oryzae (L.) and Tribolium
Castaneum (Herbst)) of Rice. Ind. Crops Prod. 2020, 143, 111849.
(19) Shahavi, M. H.; Hosseini, M.; Jahanshahi, M.; Meyer, R. L.;
Darzi, G. N. Clove Oil Nanoemulsion as an Effective Antibacterial
Agent: Taguchi Optimization Method. Desalin. Water Treat. 2016, 57
(39), 18379−18390.
(20) Al-Antary, T. M.; Belghasem, I. H.; Araj, S. E. A. Evaluation of
Eco-Friendly Lemon Oil against the Green Peach Aphid Myzus
Persicae Sulzer (Homoptera: Aphididae) Using Four Solvents.
Fresenius Environ. Bull. 2017, 25 (12), 8298−8303.
(21) Quatrin, P. M.; Verdi, C. M.; de Souza, M. E.; de Godoi, S. N.;
Klein, B.; Gundel, A.; Wagner, R.; de Almeida Vaucher, R.; Ourique, A.
F.; Santos, R. C. V. Antimicrobial and Antibiofilm Activities of
Nanoemulsions Containing Eucalyptus Globulus Oil against Pseudo-
monas Aeruginosa and Candida Spp. Microb. Pathog. 2017, 112, 230−
242.
(22) Nazarzadeh, E.; Anthonypillai, T.; Sajjadi, S. On the Growth
Mechanisms of Nanoemulsions. J. Colloid Interface Sci. 2013, 397, 154−
162.
(23) Kanwar, R.; Gradzielski, M.; Mehta, S. K. Biomimetic Solid Lipid
Nanoparticles of Sophorolipids Designed for Antileprosy Drugs. J. Phys.
Chem. B 2018, 122, 6837.
(24) Mayer, S.; Weiss, J.; McClements, D. J. Vitamin E-Enriched
Nanoemulsions Formed by Emulsion Phase Inversion: Factors
Influencing Droplet Size and Stability. J. Colloid Interface Sci. 2013,
402, 122−130.
(25) Teo, A.; Goh, K. K. T.; Wen, J.; Oey, I.; Ko, S.; Kwak, H. S.; Lee,
S. J. Physicochemical Properties of Whey Protein, Lactoferrin and
Tween 20 Stabilised Nanoemulsions: Effect of Temperature, PH and
Salt. Food Chem. 2016, 197, 297−306.
(26) Jafari, S. M.; He, Y.; Bhandari, B. Effectiveness of Encapsulating
Biopolymers to Produce Sub-Micron Emulsions by High Energy
Emulsification Techniques. Food Res. Int. 2007, 40 (7), 862−873.
(27) Fernandez, P.; André, V.; Rieger, J.; Kühnle, A. Nano-Emulsion
Formation by Emulsion Phase Inversion. Colloids Surf., A 2004, 251 (1-
3), 53−58.
(28) Sivakumar, M.; Tang, S. Y.; Tan, K. W. Cavitation Technology -
A Greener Processing Technique for the Generation of Pharmaceutical
Nanoemulsions. Ultrason. Sonochem. 2014, 21 (6), 2069−2083.
(29) Capek, I. Degradation of Kinetically-Stable o/w Emulsions. Adv.
Colloid Interface Sci. 2004, 107 (2-3), 125−155.
(30) Taylor, P. Ostwald Ripening in Emulsions: Estimation of
Solution Thermodynamics of the Disperse Phase. Adv. Colloid Interface
Sci. 2003, 106 (1-3), 261−285.
(31) Liu, W.; Sun, D.; Li, C.; Liu, Q.; Xu, J. Formation and Stability of
Paraffin Oil-in-Water Nano-Emulsions Prepared by the Emulsion
Inversion Point Method. J. Colloid Interface Sci. 2006, 303 (2), 557−
563.
https://dx.doi.org/10.1021/acsagscitech.0c00061
ACS Agric. Sci. Technol. 2021, 1, 76−88
ACS Agricultural Science & Technology pubs.acs.org/acsagscitech Article

(32) Sarheed, O.; Shouqair, D.; Ramesh, K. V. R. N. S.; Khaleel, T.;

Amin, M.; Boateng, J.; Drechsler, M. Formation of Stable Nano-
emulsions by Ultrasound-Assisted Two-Step Emulsification Process for
Topical Drug Delivery: Effect of Oil Phase Composition and Surfactant
Concentration and Loratadine as Ripening Inhibitor. Int. J. Pharm.
2020, 576, 118952.
(33) Koroleva, M.; Nagovitsina, T.; Yurtov, E. Nanoemulsions
Stabilized by Non-Ionic Surfactants: Stability and Degradation
Mechanisms. Phys. Chem. Chem. Phys. 2018, 20 (15), 10369−10377.
(34) Gupta, A.; Eral, H. B.; Hatton, T. A.; Doyle, P. S. Nanoemulsions:
Formation, Properties and Applications. Soft Matter 2016, 12 (11),
2826−2841.
(35) Ritger, P. L.; Peppas, N. A. A Simple Equation for Description of
Solute Release II. Fickian and Anomalous Release from Swellable
Devices. J. Controlled Release 1987, 5 (1), 37−42.
(36) Nuruzzaman, M.; Ren, J.; Liu, Y.; Rahman, M. M.; Shon, H. K.;
Naidu, R. Hollow Porous Silica Nanosphere with Single Large Pore
Opening for Pesticide Loading and Delivery. ACS Appl. Nano Mater.
2020, 3 (1), 105−113.

88 https://dx.doi.org/10.1021/acsagscitech.0c00061
ACS Agric. Sci. Technol. 2021, 1, 76−88