Journal of Hazardous Materials 359 (2018) 213–221

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Journal of Hazardous Materials

journal homepage: www.elsevier.com/locate/jhazmat

α-Amylase triggered carriers based on cyclodextrin anchored hollow T

mesoporous silica for enhancing insecticidal activity of avermectin against
Plutella xylostella
Amir. E. Kaziema,b, Yunhao Gaoa, Yuan Zhanga, Xueying Qina, Yanan Xiaoa, Yanhui Zhanga,
⁎
Hong Youa, Jianhong Lia, Shun Hea,
a
Hubei Insect Resources Utilization and Sustainable Pest Management Key Laboratory, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan
430070, China
b
Department of Environmental Agricultural Science, Institute of Environmental Studies and Research, Ain Shams University, Cairo 11566, Egypt

G R A P H I C A L A B S T R A C T

A R T I C LE I N FO A B S T R A C T

Keywords: α-Amylase-responsive carrier for controlled release of avermectin (AVM) was prepared based on α-cyclodextrin
Avermectin (α-CD) anchored hollow mesoporous silica (HMS) using α-CD as a capping molecule. The release of AVM was
α-Amylase response studied at different temperatures, pH values and in the presence or absence of α-amylase. The results revealed
Hollow mesoporous silica that the AVM-encapsulated controlled release formulation (AVM-CRF) has a drastic enzymatic dependence, an
Controlled release formulation
excellent encapsulation efficacy reaching 38%, and outstanding UV and thermal shielding ability. The AVM-CRF
Insecticidal activity
biological activity survey shows excellent toxicological properties against Plutella xylostella larvae, which con-
firms that α-CD caps could be uncapped enzymatically in vivo and release AVM, inducing P. xylostella larval
death. AVM-CRF has a notable capability to keep 0.6 mg L−1 AVM biologically active until 14th day with
83.33% mortality of the target insect, which was 40% higher than that of treated with AVM commercial for-
mulation. The study provides a theoretical basis for the application of pesticide reduction.

⁎
Corresponding author.
E-mail address: heshun@mail.hzau.edu.cn (S. He).

https://doi.org/10.1016/j.jhazmat.2018.07.059
Received 16 January 2018; Received in revised form 6 July 2018; Accepted 12 July 2018
Available online 20 July 2018
0304-3894/ © 2018 Elsevier B.V. All rights reserved.
A.E. Kaziem et al.
1. Introduction
Insect pests pose a risk to humankind, which has necessitated using
pesticides [1]. Recently, the use of green pesticide to minimize the risk
of ecosystem exposure has become a global trend [2]. As the bio-safety
of pesticide formulation is a vital issue in pesticide research and prac-
tical applications, many scientists have worked on developing new
controlled-release pesticide formulations to improve the pesticide for-
mulation characteristics [3,4]. Conventional release formulations are
based on adjuvants and adhesives, which are often not stimuli-re-
sponsive but instead realize release by diffusion-controlled procedures.
There is an almost total lack of efficient traditional formulations that
demonstrate selective action in the presence of target insects. As a
promising alternative, surface-functionalized hollow mesoporous silica
(HMS) offers exceptional characteristics as a drug carrier, including, for
instance, high stability, excellent biocompatibility, high loading effi-
ciency, safety of untargeted biota from any accidental release, simple
fabrication, and the ability to attach molecules on exterior surfaces that
can work as stoppers to control the release of entrapped molecules
[5–10]. Many researchers have taken revolutionary steps in pesticide
formulation, with numerous substances being involved, to realize a
green formulation capable of achieving a targeted purpose without
harming surrounding organisms. Controlled pesticide-release systems
such as solid-lipid nanoparticles [11,12], polymeric nanospheres
[13,14], nanosized metals and metal oxides [15,16], and layered
double hydroxides and clays [17,18] are reported previously. All these
formulations achieve great loading capacity and release behavior but
do not provide control over where the pesticide release will occur.
The development of gated stimuli-responsive HMS is a new research
area that transports molecular and supramolecular ideas to the frontiers
of controlled-release science [19,20]. A large quantity of controlled-
release formulations are encouraged by bio-gates, which utilize mo-
vable stoppers triggered by a precise stimulus. To date, scientists have
demonstrated several mesoporous silica-based formulations with con-
trolled-release properties with diverse gated structures, which in most
of the cases use stimulators to uncap the pores such as light [21,22], pH
[23], redox potential [24,25], and enzymes [25,26]. Additional systems
include pseudorotaxanes [27], carboxylates [28], and complexes such
as cucurbit[6]uril [29], cucurbit[7]uril [30], and cyclodextrins [31].
Even with these many instances, the methodologies for utilizing gated
HMS for the expansion of real delivery systems are still in their pre-
liminary stages.
To realize the benefit of carbohydrate digestion by linking their
digestion procedure to controlled-release pesticide formulation tech-
nology, α-cyclodextrin (α-CD) can be utilized. α-CD is a member of the
cyclic oligosaccharide family; it is composed of α-(1,4)-linked gluco-
pyranose subunits that can be hydrolyzed enzymatically by the α-
amylase enzyme. Enzymes are the assimilation keys, which exist in any
digestive system; α-amylase, which is present in the salivary glands and
midgut of chewing mouthparts larvae [32,33], is the critical factor of
the study.
Avermectin (AVM) is a widely-used insecticide, acaricide, and ne-
maticide. AVM is a combination of avermectins, containing more than
80% avermectin B1a and less than 20% avermectin B1b. AVM has
stomach toxicity activity that can influence keeping the γ-aminobutyric
acid-gated chloride channels, glutamate-gated chloride channel, and
other chlorine channels open in insect muscle membranes, which leads
to interdicting the synaptic transmission from internuncial neurons to
motor nerve cells and the synaptosome peripheral nerve conduction
between the neuromuscular system [34]. Numerous delivery systems
have been prepared to enhance both AVM stability under UV light and
its dissolvability in water, such as lignocellulosic matrices [35], porous
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Journal of Hazardous Materials 359 (2018) 213–221
hollow silica with various shell thicknesses [36,37], starch micro-
capsules [38], porous acrylic resin [39], silica microcapsules [40], cy-
anobacteria [41], and cellulose acetate ultrafine fibers [42]. Although
this controlled release depends on sustained release, few research stu-
dies have paid attention to the interaction between the formulation and
insect enzymes, especially for release of the packed pesticide in the
larval midgut.
Herein, we report the fabrication of enzyme triggered system based
on cyclodextrin anchored hollow mesoporous silica and the release
profile of the system under different temperatures, different pH values,
in the presence or absence of α-amylase enzyme as well as an in vivo
experiment on Plutella xylostella to estimate the AVM-HMS toxicological
activity. We focus on the utilization of the midgut α-amylase enzyme as
the AVM-CRF biological release trigger. To date, examples of biomo-
lecule use in capping or uncapping protocols in pesticide formulations
have not been reported.
2. Material and methods
2.1. Materials
2,2′-azobis(2-methylpropionamidine) di-hydrochloride (V-
50, > 97.0%), styrene (> 99.0%, stabilized with TBC), hexadecyl-
trimethylammonium bromide (CTAB, > 99.0%), ammonium hydroxide
solution (28 wt%), and polyvinylpyrrolidone (K-30, > 99.5%) were
obtained from Aladdin Reagent. Co., Ltd (Shanghai, China). N-phenyl
aminopropyltrimethoxysilane (PhAPTMS 96%) and α-cyclodextrin (α-
CD 98%) were obtained from Heowns Biochem Technologies, LLC
(Tianjin, China). α-amylase enzyme (enzyme activity is
50 μmol mg−1 min−1) was purchased Sigma Aldrich Inc. Methanol
(> 99.9%), toluene (> 99.5%), acetone (99.5%), ethanol (99.9%) and
tetraethyl orthosilicate (TEOS > 99.9%) were obtained from
Sinopharm Chemical Reagent Co., Ltd (Beijing, China). All chemicals
were used without additional purification. Deionized water was gen-
erated with a Sartorius Stedim arium pro-Ultrapure Water System
(Sartorius, Germany).
2.2. Experimental
2.2.1. Synthesis of functionalized HMS
The preparation of AVM-CRF starts with the as-reported synthesis of
the polystyrene latex (PSL) template [43], with little modifications. PSL
template was prepared by emulsion polymerization as follows, K-30
was dissolved in water under vigorous stirring in a three-necked flask;
styrene was added dropwise, and the system was kept stirring under
nitrogen purging for 30 min to form an emulsion. The emulsion was
heated to 70 °C in an oil bath. The polymerization was initiated by
injecting the V-50 aqueous solution into the reaction, and then, the
emulsion continued polymerization for 24 h under nitrogen to form the
PS latex.
For HMS synthesis, CTAB was dissolved in a solution of water,
ethanol, and ammonium hydroxide. An exact amount of PSL was added
dropwise to the solution. This was followed by sonication for 120 min
and then 30 min magnetic stirring followed by the dropwise addition of
TEOS. The mixture was then magnetically stirred at 40 °C for 48 h. By
centrifugation, the PSL plated with silica was gathered, which was then
washed by ethanol thrice and dried under vacuum at room temperature
(RT) for 24 h. The PSL template was removed at 600 °C for 8 h in air. To
obtain a phenylamine surface-functionalized HMS, the HMS was sus-
pended in dried toluene with PhAPTMS and kept at reflux under N2 for
24 h. The gathered surface-modified HMS was washed with dry toluene
and methanol and then dried under vacuum. AVM loading was
A.E. Kaziem et al.
performed by immersing PhAP-HMS in AVM solution and then main-
taining stirring for 24 h. The AVM-loaded PhAP-HMS was gathered,
followed by washing and drying under vacuum at RT. To obtain sealed
HMS, AVM-loaded PhAP-HMS was placed in a water solution con-
taining an excess amount of α-CD, which was then sonicated and
magnetically stirred for three days at RT. Finally, the precipitate was
collected by centrifugation and washed with water thrice until no dye
was detected in the washing solution.
2.2.2. Characterization apparatus
A JEM-2100 F transmission electron microscope (TEM, JEOL,
Japan) and SU8200 scanning electron microscope (SEM, Hitachi,
Japan) were used to observe and characterize the HMS morphology and
structures. Dynamic light scattering (DLS) and zeta potential was ana-
lyzed by a Zetasizer nano ZS particle size instrument (Malvern Co.,
U.K.). Additionally, a Fourier transform infrared spectroscopy (FT-IR,
Nexus 470, USA) VORTEX 70 instrument was conducted to identify the
chemical grafting processes of HMS. Thermogravimetric analysis (TGA,
Netzsch DSC200PC/TG209C, Germany) explored the HMS weight loss
in air. The BET surface area and the pore size distributions of the
synthesized HMS and α-CD-HMS were measured by N2 adsorption-
desorption isotherms, which were conducted at 77 K on a Micromeritics
ASAP 2460 analyzer (Micromeritics Instrument Corporation, USA)
under continuous adsorption conditions. High-performance liquid
chromatography (HPLC) (Shimadzu, Japan) analyzed the released AVM
from AVM-CRF, with the following conditions: chromatographic
column kromasil ODS C18 (250 mm × 4.6 mm, 5.0 μm). A guard
column (4 mm × 3 mm) was pre-equilibrated to the C18 column. All
solvents were pre-filtered with a 0.45 mm filter membrane. For AVM,
the mobile phase was a mixture of water and methanol (8:92, v/v) with
a flow rate of 1 mL·min−1. The detector was a UV detector, and the
detection wavelength was 244 nm. The injection volume was 20 μL for
all samples and standards.
2.2.3. Controlled release kinetics
The AVM release from AVM-CRF under different variables such as
different pH values, temperatures, and the presence of α-amylase en-
zyme was investigated. For the different investigations, 0.2 g of AVM-
CRF was dispersed in 500 mL of acetone-H2O medium (30:70, v/v) at
100 rpm stirring speed in a RC1208D dissolution tester (Tianjin, China).
Scheme 1. The synthesis steps of α-CD-AVM-PhAP-HMS CRF.
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Journal of Hazardous Materials 359 (2018) 213–221
The cumulative release of the encapsulated AVM from the CRF in the
release medium was determined. Samples (2.0 mL) were withdrawn at
various times from the release suspension in the dissolution tester,
followed by centrifugation at 10 000 rpm to obtain 50 μL supernatant
samples removed from the CRF particles for HPLC analysis. The re-
maining mix was returned to the tester flask. All quantitative trials were
conducted thrice. The mean values with standard deviations were cal-
culated.
2.2.4. Durability under UV stress and different temperature
The stability of the AVM-CRF under different temperatures in
comparison with AVM technical grade was investigated. Each sample
was (0.2 g) packed in a dark Pyrex tube and stored at 45 °C, 55 °C, or
65 °C for 60 days to determine the effects of different temperatures on
the loaded AVM degradation. The active ingredient (AI) alterations in
all samples were analyzed by HPLC. The AVM-CRF UV-shielding
properties were examined and compared with those of AVM technical
grade. Each sample (0.2 g), both AVM-CRF and technical grade, was
mixed at RT with 500 mL of an acetone-H2O mixture (30:70, v/v) and
then transferred to a 1000 mL cylindrical reaction vessel fitted with a
magnetic stirrer, which was then exposed to a 36 W germicidal lamp
(254 nm) at 20 cm distance. Two milliliters of the release solution was
withdrawn with a digital SLP cell disruptor (SLPt Time, Emerson
Electric Co., USA) at different periods. The aliquot was then cen-
trifuged, and the gained supernatant was used for HPLC analysis to
determine the changes of AI in AVM-CRF compared with those of the
control AVM technical grade.
2.2.5. Bioassay
To investigate the AVM-CRF toxicological effect in vivo, we tested
the biological activity of AVM-CRF compared with that of the AVM
commercial formulation (AVM-CF) EC 1.8% on Brassica oleracea against
the 3rd instar larvae of P. xylostella, which were reared and nurtured on
a diet of freshly grown B. oleracea seedlings. The bred larvae were kept
at relative humidity 70 ± 10%, 25 ± 1 °C, and 16L:8D. B. oleracea
was cultivated to at least 7 leaves in greenhouse without applying any
pesticides. Stock solutions at 20.0 mg L−1 of blank HMS, AVM-HMS,
AVM-CF, and AVM-CRF were prepared by dispersing the pesticide
formulation in water. After the plants had been sprayed with the as-
signed formulation, the plant leaf discs (6.5 cm diameter) were
A.E. Kaziem et al. Journal of Hazardous Materials 359 (2018) 213–221

collected at sequential times (0, 1, 3, 5, 7, 10, and 14 days) to feed the

insects. Three replicates of 10 3rd instar larvae were tested for each
concentration. The mortality was assessed 72 h after exposure to AVM.

2.2.6. Statistical analysis

Statistical analysis was performed using SPSS 24.0 statistical ana-
lysis software (SPSS, Chicago, IL, USA) and analyzed by one-way ana-
lysis of variance technique [44]. All of the data generated from three
independent experiments were presented as the mean ± SD. LSD tests
were performed to compare the treatments difference. The value of
p < 0.05 was considered to be significant different.

3. Results and discussions

3.1. Synthesis and characterization of AVM-CRF

In order to avoid complex synthetic routes to get AVM, we selected Fig. 2. FT-IR spectrum of Blank HMS (A), PhAP-HMS (B), AVM-PhAP-HMS (C),
commercially available α-CD as the capping agent. In acidic medium, and α-CD-AVM-PhAP-HMS (D). KBr was used as a background.
ionized N-alkylaniline of PhAPTMS exhibited a much weaker binding to
β-CD than that with α-CD due to their enhanced hydrophilic character
3.1.1. TEM and SEM observations
[45]. In order to prevent the pesticide released in advance, we chose α-
The surface morphology of the samples were examined by TEM and
CD that is more stable in complexation with N-alkylaniline. Scheme 1
SEM (Fig. 1). The HMS exhibited a uniform structure with spherical
shows the formation of α-CD-AVM-PhAP-HMS.
shapes. TEM showed the HMS is hollow spheres, which were obtained

Fig. 1. SEM images of HMS (a) and α-CD capped HMS (b), TEM images of HMS (c) and α-CD capped HMS (d), HR-TEM images of mesoporous channels of HMS (e)
and mesoporous channels of HMS after α-CD capping (f). (All the observed samples on SEM have been coated by gold dust).

216
A.E. Kaziem et al.
after the removal of PSL template. The low-magnification TEM images
display excellent particle symmetry and demonstrated that all the silica
particles had a hollow profile. The HR-TEM images show the meso-
porous channels’ shape before and after anchored with α-CD, which
confirm the mesoporous channels’ shape change after α-CD anchored.
The HMS average exterior and interior diameters were about 400 nm
and 380 nm respectively. The HMS shell width was about 28 nm.
3.1.2. FT-IR
The samples were qualitatively confirmed by FT-IR spectra, which
are shown in Fig. 2. For the HMS spectrum, the absorption bands in the
range of 3750–3000 cm−1 are due to silanol stretching vibrations, and
the sharp peaks at 1084 cm−1 can be assigned to the stretching vibra-
tions of the mesoporous structure (Si–O–Si) (Fig. 2A). However, during
the surface functionalization, the surface hydroxyl groups react with
the PhAPTMS, which reduces the (eOH) group. Fig. 2B shows a re-
duction in the hydroxyl groups, which suggests an attachment me-
chanism involving a reaction between the silanol groups and PhAPTMS.
Furthermore, two bands assigned to (eCH2e) stretching were observed
at 2933 cm−1 and 2871 cm−1 due to the methyl groups introduced
during functionalization. In addition, the (eNHe) stretching band can
be observed at 3375 cm−1. Furthermore, the vibration peak of aromatic
hydrocarbons (eC = Ce) appears at 1604 cm−1 and in an extended
band from 1499 cm−1 to 1508 cm-1. AVM-PhAP-HMS (Fig. 2C) shows a
much stronger absorption band at 3448 cm−1 owing to AVM (eOH)
stretching; it also shows higher absorption at 2967 cm−1 and
1389 cm−1 due to the clear rise of AVM methyl group (eCH3) asym-
metrical stretch content. Moreover, the AVM aliphatic ketone group
(C]O) absorption band appears at 1735 cm−1, and the aromatic hy-
drocarbon C]C stretch absorption band appears at 1459 cm−1, de-
monstrating that AVM has encapsulated into the HMS. α-CD-AVM-
Fig. 3. Nitrogen adsorption-desorption isotherms for blank HMS, and α-CD capped HMS, Insets: Pore size distribution of each HMS, and α-CD-HMS (A). Size
distribution of HMS (B). TGA curves for blank HMS, α-CD-PhAP-HMS, and α-CD-AVM-PhAP-HMS (C).
217
Journal of Hazardous Materials 359 (2018) 213–221
PhAP-HMS (Fig. 2D) shows wide peak shape extension at 3387 cm−1,
which is attributed to α-CD (eOH). (Supplementary data Fig. S1 and
S2)
3.1.3. Surface area and pore size distributions (BET analysis)
N2 adsorption-desorption isotherm of the blank HMS and α-CD
capped HMS, Fig. 3A, displays a type IV isotherm with a hysteresis loop,
which clearly proves its porous feature possessing cylindrical pores
geometry with a great degree of pores size uniformity. The adsorption
step ascribed to the mesopores and the pores texture is preserved an
appreciable decrease in the N2 volume adsorbed (BJH mesopore vo-
lume = 0.31 cm3 g−1). The blank HMS surface area was measured
(0.654 ☓ 103 m2 g−1). The surface area of solid α-CD capped HMS was
reduced about 70%, when compared to that presented by blank HMS,
due to the external grafting of α-CD. For blank HMS, the BJH pore size
distribution shows a maximum at 3.13 nm on the border between me-
sopores and micropores. On the other hand, the adsorption step at high
relative pressure (P/P0 > 0.8) which appears in the isotherm of HMS,
and α-CD capped HMS can be estimated. For blank HMS, the BJH N2
adsorption was 0.46 cm3 g−1, whereas α-CD capped HMS was
0.14 cm3 g−1. These data support the fact that the α-CD caps are spe-
cifically incorporated on the pores rims of the ordered mesopores. BET
specific surface values, pore volume, and pore size calculated from the
N2 adsorption-desorption isotherms for blank HMS and α-CD capped
HMS are listed in Table 1.
3.1.4. Dynamic light scattering (DLS) and zeta potential
A Zetasizer instrument was conducted to investigate the particle size
distribution and zeta potential. The average size was about 400 nm
(Fig. 3B). This value is consistent with TEM result. The zeta-potentials
of the samples were measured by Zetasizer. Blank HMS showed
A.E. Kaziem et al. Journal of Hazardous Materials 359 (2018) 213–221

Table 1 observed, with the slight increase in weight loss over the blank HMS
Properties of blank HMS and α-CD capped HMS calculated from N2 adsorption- due to the α-CD existence. AVM-CRF weight was distinctly diminished,
desorption isotherms. 41.2%, owing to the AVM loaded into the HMS internal sphere: the
Sample SBET (m2·g−1) BJH pore size BJH Pore volume accurately loaded AVM amount into the HMS, encapsulation efficacy
(nm) (cm3·g−1) (EE), was 38% of the total HMS weight. (Supplementary data Fig. S3
and S4)
Blank HMS 0.654 ☓ 103 3.13 0.31
α-CD capped HMS 0.191 ☓ 103 1.74 0.02
3.2. Controlled release kinetics

negative charge, the zeta potential was −30 mV. In the case of phenyl
group-modified HMS, the Ph-HMS showed negative charges −23 mV.
The zeta potential of the Ph-HMS was less negative than that in blank
HMS, which indicated that the phenyl groups decreased the effect of the
(−OH) group on the HMS surface. For α-CD-Ph-HMS, the zeta potential
was −36 mV which was smaller than that of Ph-HMS. This may be due
to the introduction of α-CD, resulting in an increase in the number of
hydroxyl groups. Thus, the surface modification of HMS was success-
fully achieved. The results agreed with the previous studies [46,47].
3.1.5. Drug loading efficiency (DLE)
To inspect the collapsed weight of α-CD-AVM-PhAP-HMS, α-CD-
PhAP-HMS, and blank HMS, thermogravimetric analysis TGA was used
(Fig. 3C). The TGA results demonstrated that the α-CD and AVM de-
composition started at 245 °C and 241 °C respectively. Due to vapor
volatilization, 4.96% of the total weight of blank HMS was lost. In
contrast, 7.91% loss from the total weight of α-CD-PhAP-HMS was
In order to examine the release profile of the AVM-CRF compared
with the HMS uncapped with α-CD at neutral conditions, the release
medium was adjusted to pH 7, 100 rpm stirring rate, and RT. Fig. 4A
shows the AVM-CRF cumulative release was 5.88% while the AVM-
HMS was 71.54% on the 17th day. The release profiles of AVM-CRF had
a low initial release rate (approximately 6%), which agreed with the
previous studies [25,26].
Fig. 4B displays the AVM release behaviors from AVM-CRF under
pH values 5, 7, and 10. At pH 5, the cumulative release of AVM in-
creased from 4.19% at the 4th day to 6.13% at the 17th day; the release
rose from 3.98% to 5.88% from the 4th day to the 17th day at pH 7.
However, the cumulative release rose from 18.22% to 40.24% at pH 10.
This result is due to the instability of Si-O-Si bonding under strong al-
kaline condition, leading to disintegrate the hollow mesoporous struc-
ture. The results agreed with the previous studies [48,49].
Fig. 4C displays the cumulative release of AVM from AVM-CRF. The
cumulative release was 3.98%, 4.15%, and 5.59% at the 4th day for

Fig. 4. Release profile of AVM-CRF compared with AVM-HMS uncapped with α-CD under neutral conditions (mean ± SD, n = 3 for each sample) (A). Cumulative
release profile of AVM-CRF under pH 5, pH 7, and pH 10 at RT (mean ± SD, n = 3 for each sample) (B). Release behavior of AVM-CRF at 25 °C, 35 °C, and 45 °C. At
pH 7 and 100-rpm (mean ± SD, n = 3 for each sample) (C). The influence of α-amylase enzyme (0.65 mg mL−1) addition in the 4th day at RT, pH 7, and 100-rpm on
AVM-CRF unsealing and the cumulative release: AVM-CRF release without enzyme, AVM-CRF release with the enzyme. (mean ± SD, n = 3 for each sample) (D).

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A.E. Kaziem et al. Journal of Hazardous Materials 359 (2018) 213–221

25 °C, 35 °C, and 45 °C respectively., The cumulative release were less 0.1 mg L−1 and 0.2 mg L−1 AVM-CF, AVM-HMS, and blank HMS. The
than 10% for 25 °C, 35 °C, and 45 °C until 17th day. These results at- mortality at Day 1, for blank HMS, was the same as that of Day 0 for all
tributed to the higher the temperature, the more intense the motion of concentrations, except 0.1 mg L−1 mortality which was 6.67%. The
liquid molecules, which caused a smoother and faster AVM release from AVM-CRF mortality at Day 3 decreased for all concentrations but was
the AVM-CRF at a higher temperature [50]. still higher than that of AVM-CF, AVM-HMS, and blank HMS at the
The AVM-CRF release behaviors in the presence or absence of the α- same day. At Day 5 (Supplementary data Table S1), mortality of
amylase enzyme were investigated while keeping the pH value (pH 7) 0.05 mg L-1 AVM-CF decreased in varying degrees for all concentra-
constant at a stirring speed of 100 rpm. Fig. 4D displays the influence of tions, which was ascribed to the surrounding degradation influences
α-amylase existence on the cumulative release of AVM-CRF. The release compared with the same AVM-CRF concentration, which decreased by
behaviors were similar until 4th day before adding the α-amylase so- 6.66% compared with the mortality of Day 3, attributed to the CRF
lution (0.65 mg mL−1). The release behavior without enzyme shifted shielding capability. From Day 7, mortality of AVM-CRF was significant
from 3.98% to 5.88% from the 4th day to the 17th day, while the release higher than that of AVM-CF under all treatment concentrations. At Day
behavior in the presence of the enzyme shifted from 3.93% to 41.62% 14, the mortality for AVM-CRF 0.6 mg L−1 was still higher than 80%,
in the same elapsed time. The result validated the theory of selectively unlike that of the AVM-CF, AVM-HMS, and blank HMS 0.6 mg L−1,
unsealing the CRF in the presence of α-amylase enzyme by the rupture which decreased to 43.3%, 53.33%, and 3.33% respectively. The LC50
of the α-cyclodextrin α-1,4 bonds between glucose monomers. α- of AVM-CRF against P. xylostella is only about 41.0% to 57.4% of the
Amylase hydrolyzes the alpha bonds of large alpha-linked poly- AVM-CF until Day 5. While for Day 10 and 14, less than 30%, which
saccharides, such as α-CD, yielding glucose monomers. In insects, α- means it can reduce pesticide use by about 50%–70%.
amylase exists in salivary glands and the midgut. This serves as the The efficacy of pesticides progressively decreases with time. As a
critical key to release the AVM from the AVM-CRF. concept in pesticide nano formulation technology, AVM-CRF aims to
prolong biological effectiveness half-life to the greatest extent possible.
3.3. Study of thermal and UV stability With the intention of determining the AVM effectiveness life in AVM-
CRF, the AVM-CRF 0.6 mg L−1 experimented plants were kept after Day
The AVM-CRF and technical pesticide were stored for 60 days at 14 for another 30 days with leaf samples withdrawn at Day 16, 20, 25,
40 °C, 50 °C, and 60 °C. HPLC was used to detect changes in the AI with 35, and 44. AVM remained active, realizing 53.33% mortality at Day
the abovementioned method. Table 2 shows that the technical AVM 25, 36.67% mortality at Day 35 and 6.67% mortality at Day 44.
decomposed more rapidly and easily under high temperatures than the
AVM encapsulated AVM-CRF. For UV photolysis, the technical AVM 4. Conclusions
was easier to photolyze than of AVM inAVM-CRF. The technical AVM
exhibited more than 98% decomposition after 3 h of UV radiation. After In summary, we developed an enzyme-response AVM-CRF con-
24 h of UV radiation, however, less than 9% of AVM in the CRF was trolled pesticide release system through the interactions between α-CD
photolyzed. The revealed data confirmed that the prepared CRF can and PhAP-HMS forming immobilized stoppers on the surface of HMS
protect AVM from photolysis, which demonstrated the extraordinary pore. The results showed that AVM-CRF had an excellent loading ability
UV-shielding property of the AVM-CRF. for avermectin (about 38% w/w). AVM-CRF could protect avermectin
against photolysis effectively. AVM-CRF showed excellent controlled
3.4. Bioassay release properties, and it can accelerate the release of avermectin in the
presence of α-amylase. The bioassay of AVM-CRF showed excellent
The 3rd instar larvae of P. xylostella was selected as a model insect in biological activities against P. xylostella larvae, which confirms that α-
order to assess the obtained AVM-CRF biological activity. The LC90 of CD caps could be uncapped enzymatically in vivo and release the pes-
the AVM technical was calculated by SPSS statistical software, and the ticides, inducing P. xylostella larval death. The toxicity of AVM-CRF
resulting LC90 was 0.5391 mg L−1. The AVM-CRF biological activity against P. xylostella is significant stronger than that of the same con-
was compared with that of the AVM commercial formulation EC 1.8% centration of AVM-CF after spraying. In agricultural applications, pes-
(AVM-CF), AVM loaded HMS without capping by α-CD, and blank ticides usage can be reduced by 50–70% by using AVM-CRF reported in
HMS. Five concentrations were set, 0.05, 0.1, 0.2, 0.4, and 0.6 mg L−1, the study.
for blank HMS, AVM-HMS, AVM-CRF, and AVM-CF. Seven pickings
were defined, Day 0, Day 1, Day 3, Day 5, Day 7, Day 10, and Day 14, to
Acknowledgements
evaluate and compare the efficacy of AVM-CRF, AVM-HMS, blank HMS,
and AVM-CF. Fig. 5 and Table S1 (Supplementary data) present the
This work was supported by National Key R&D Program of China
derived biological activity results.
(2017YFD020030803), Key Projects of Hubei Province Technological
Toxicity of AVM-CRF, AVM-CF, and AVM-CRF against P. xylostella
are presented in Table 3. It was observed that the surviving insects’
Table 2
activity in the AVM-CRF treatment trials was very weak, which was
The stability of AVM-CRF under UV-radiation and different temperatures.
attributed to AVM interdiction of the synaptic transmission from the
internuncial neurons to the motor nerve cells. The mortality was high at Tests at different conditions Decomposition rate (%)
Day 0 for all AVM-CRF treatments, realizing 36.67% mortality for
Avermectin technical AVM-CRF
0.05 mg L−1 up to 100% mortality for 0.6 mg L−1, compared with
AVM-CF, AVM-HMS and blank HMS, which had 13.33% mortality for Irradiation at 254 nm for 1h 50.85 2.19
0.05 mg L−1 up to 100% at 0.6 mg L−1, 23.33% mortality for Irradiation at 254 nm for 2h 88.66 2.97
0.05 mg L−1 up to 100% at 0.6 mg L−1, and 0 mortality from Irradiation at 254 nm for 3h 98.03 3.65
Irradiation at 254 nm for 4h 100 4.41
0.05 mg L−1 to 0.6 mg L−1, respectively. For Day 1 (Supplementary Irradiation at 254 nm for 6h 100 5.23
data Table S1), the mortality was slightly different for 0.2 mg L−1 and Irradiation at 254 nm for 12 h 100 6.18
0.4 mg L−1 treatments, which decreased by 3.33% and 6.67% from Irradiation at 254 nm for 18 h 100 7.26
their Day 0 AVM-CRF values; the AVM-CF 0.2 mg L−1 and 0.4 mg L−1 Irradiation at 254 nm for 24 h 100 8.21
Storage 60 days at 40 °C 5.01 1.09
values each decreased by 13.33% from their Day 0 mortality. For AVM-
Storage 60 days at 50 °C 9.85 2.18
CRF 0.1 mg L−1 and 0.2 mg L−1, the mortalities were 53.33% and Storage 60 days at 60 °C 17.14 3.94
83.33% at Day 1, respectively, which were much higher than that of

219
A.E. Kaziem et al. Journal of Hazardous Materials 359 (2018) 213–221

Fig. 5. Mortality of different concentrations of AVM-CRF, AVM-HMS, AVM-CF, and blank HMS against P. xylostella. Statistically significant differences are indicated
by *P ＜ 0.05 as compared with AVM-CF.

Table 3
Toxicity of AVM-CRF, AVM-CF, and AVM-CRF against P. xylostella.
Time after spraying (day) LC50 (mg·L−1, 95% fiducial limit) LC90 (mg·L−1) Slope (mean ± SE) Chi-square value

AVM-HMS Day 0 0.108 (0.072-0.162) 0.37 2.39 ± 0.08 0.94

Day 1 0.141 (0.089–0.223) 0.65 1.91 ± 0.10 0.75
Day 3 0.183 (0.117–0.286) 0.81 1.98 ± 0.09 0.86
Day 5 0.234 (0.145–0.377) 1.17 1.83 ± 0.10 0.75
Day 7 0.391 (0.228–0.670) 2.33 1.65 ± 0.11 0.93
Day 10 0.502 (0.294–0.858) 2.77 1.73 ± 0.11 0.46
Day 14 0.627 (0.359–1.097) 6.7 1.72 ± 0.12 0.55
AVM-CF Day 0 0.136 (0.094–0.198) 0.43 2.61 ± 0.08 0.20
Day 1 0.175 (0.118–0.261) 0.63 2.31 ± 0.08 1.26
Day 3 0.220 (0.147–0.327) 0.81 2.31 ± 0.08 3.23
Day 5 0.329 (0.218–0.495) 1.15 2.30 ± 0.09 1.14
Day 7 0.544 (0.321–0.921) 2.56 1.80 ± 0.11 0.59
Day 10 0.691 (0.401–1.193) 2.28 1.82 ± 0.12 0.20
Day 14 0.938 (0.501–1.756) 3.41 1.62 ± 0.13 0.12
AVM-CRF Day 0 0.078 (0.118–0.051) 0.27 2.46 ± 0.09 0.92
Day 1 0.081 (0.051–0.130) 0.35 2.04 ± 0.10 0.44
Day 3 0.100 (0.059–0.169) 0.58 1.70 ± 0.11 0.44
Day 5 0.135 (0.089–0.203) 0.57 2.26 ± 0.09 2.90
Day 7 0.166 (0.107–0.258) 0.79 2.02 ± 0.09 4.06
Day 10 0.204 (0.125–0.334) 1.10 1.76 ± 0.10 1.69
Day 14 0.222 (0.137–0.357) 1.12 1.83 ± 0.10 1.62

Innovation (2016ABA104), National Natural Science Foundation of online version, at https://doi.org/10.1016/j.jhazmat.2018.07.059.

China (31601654), the Special Fund for Agro-Scientific Research in the
Public Interest (201303027), and Fundamental Research Funds for the
Central Universities (2662016PY047).
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
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