Octahydrogenated retinoic acid-conjugated glycol chitosan nanoparticles as a novel carrier of azadirachtin: Synthesis, characterization, and in vitro evaluation

Article abstract of DOI:10.1002/pola.26801

Environment-friendly and controlled release formulation is highly promising for reducing environmental pollution and achieving the most effective utilization of pesticides. As a novel green nanocarrier of pesticides, amphiphilic self-assembled nanoparticles were prepared by chemical conjugation of octahydrogenated retinoic acid (OR) to the backbone of glycol chitosan (GC). In aqueous media, the synthesized OR-GC conjugates formed nanosized particles with a diameter of 257 nm. Hydrophobic azadirachtin (AZA) was efficiently loaded into the OR-GC nanoparticles at a feed weight ratio of up to 1:4 using a simple dialysis method, the maximum drug-loading efficiency of which was 74%. AZA-OR-GC (25 wt %) nanoparticles also showed sustained release of the incorporated AZA (65% of the loaded dose was released in 7 days at 27°C in phosphate-buffered saline; pH 7.2). Cytotoxicity tests and cell cycle arrest assays confirmed that OR-GC exhibits good biocompatibility; AZA-OR-GC (25 wt %) nanoparticles also showed favorable inhibition of cell proliferation in Sl-1 cells compared with free AZA in organic solvents. Overall, controlled release AZA-OR-GC may be a promising environment-friendly formulation for integrated pest management. Copyright

Full text of DOI:10.1002/pola.26801

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Octahydrogenated Retinoic Acid-Conjugated Glycol Chitosan
Nanoparticles as a Novel Carrier of Azadirachtin: Synthesis,
Characterization, and In Vitro Evaluation
Wei Lu,1,2 Meng-Ling Lu,1,2 Qing-Peng Zhang,2,3 Yong-Qing Tian,1,2
Zhi-Xiang Zhang,2,3 Han-Hong Xu1,2
1State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, South China Agricultural University,
Guangzhou 510642, China
2Key Laboratory of Natural Pesticide and Chemical Biology of the Ministry of Education, South China Agricultural University,
Guangzhou 510642, China
3Laboratory of Insect Toxicology, South China Agricultural University, Guangzhou 510642, China
Correspondence to: H. H. Xu (E-mail: hhxu@scau.edu.cn) or Z. X. Zhang (E-mail: zdsys@scau.edu.cn)
Received 2 May 2013; accepted 30 May 2013; published online 26 June 2013
DOI: 10.1002/pola.26801
ABSTRACT: Environment-friendly and controlled release formu-
lation is highly promising for reducing environmental pollu-
tion and achieving the most effective utilization of pesticides.
As a novel “green” nanocarrier of pesticides, amphiphilic self-
assembled nanoparticles were prepared by chemical conjuga-
tion of octahydrogenated retinoic acid (OR) to the backbone
of glycol chitosan (GC). In aqueous media, the synthesized
OR-GC conjugates formed nanosized particles with a diameter
of 257 nm. Hydrophobic azadirachtin (AZA) was efficiently
loaded into the OR-GC nanoparticles at a feed weight ratio of
up to 1:4 using a simple dialysis method, the maximum drug-
loading efficiency of which was 74%. AZA-OR-GC (25 wt %)
nanoparticles also showed sustained release of the
incorporated AZA (65% of the loaded dose was released in 7
days at 27 ꢀC in phosphate-buffered saline; pH 7.2). Cytotoxic-
ity tests and cell cycle arrest assays confirmed that OR-GC
exhibits good biocompatibility; AZA-OR-GC (25 wt %) nano-
particles also showed favorable inhibition of cell proliferation
in Sl-1 cells compared with free AZA in organic solvents.
Overall, controlled release AZA-OR-GC may be a promising
environment-friendly formulation for integrated pest manage-
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2013 Wiley Periodicals, Inc. J. Polym. Sci., Part A:
Polym. Chem. 2013, 51, 3932–3940
KEYWORDS: azadirachtin; glycol chitosan; in vitro evaluation;
nanocarrier; self-assembled nanoparticles; Sl-1 cells
health. Therefore, the development of pesticide formulations
has paid significant attention to environmental safety, reduc-
tions in organic solvent usage, and improving the bioavaila-
bility and persistence of the active ingredient.9 Many studies
have focused on developing novel environment-friendly and
controlled release aqueous solutions.10
INTRODUCTION Azadirachtin (AZA) is a potent tetranortriter-
penoid botanical insecticide extracted from the seeds of
neem tree (Azadirachta indica). It has demonstrated signifi-
cant advantages in integrated pest management (IPM)
against a wide range of insect pests, such as a broad spec-
trum of activity, no known insecticide resistance mecha-
nisms, new mode of action with possible multiple sites of
attack, compatibility with many commercial insecticides, fun-
gicides, and other biological agents, and minimal impact on
nontarget organisms.1–4 AZA is a hydrophobic pesticide with
low water solubility (0.26 mg mL21, 20 ꢀC).5,6 Sunlight or
UV can quickly degrade AZA because its molecules contain
unstable groups, such as olefinic bond, epoxy, and enol struc-
tures.7,8 However, traditional formulations of emulsifiable
AZA concentrates and dusts employ a large amount of
organic reagents, which are not conducive to reducing envi-
ronmental pollution, promoting food safety and human
Nanosized drug carriers, such as micelles, nanoparticles,
polymer-drug conjugates, and stealth liposomes, have been
investigated in attempts to enhance drug efficacy and bioa-
vailability.11,12 Polymeric amphiphiles consisting of hydro-
philic and hydrophobic segments have received much
attention because they can form self-assembled nanoparticles
and exhibit good physicochemical and controlled release
characteristics. In the aqueous phase, the hydrophobic cores
of polymeric nanoparticles are surrounded by hydrophilic
outer shells. Thus, self-assembled polymeric nanoparticles
Additional Supporting Information may be found in the online version of this article.
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have been recognized as promising drug carriers because
their inner core can serve as reservoirs for hydrophobic
drugs.13,14
>10 MX). All other chemicals were purchased from Sino-
pharm Chemical Reagent, which were analytical grade and
used without further purification.
Significant research attention has recently been paid to the
preparation of biodegradable and nontoxic polymeric amphi-
philes based on natural biomaterials, such as chitosan.9 Chi-
tosan and its derivatives have specific structures and
physicochemical properties, leading to excellent biocompati-
bility, biodegradability, low immunogenicity, and biological
activities.15–17 However, chitosan is soluble only in acidic
aqueous solutions below pH 6.5.18 Most chitosan-based self-
aggregates precipitate within a few days in biological solu-
tion (pH 7.4), which restricts the medical application of chi-
tosan and its derivatives in drug delivery systems.19
However, glycol chitosan (GC) is a novel chitosan derivative
and carrier of drugs because of its solubility in water at all
pH values and biocompatibility.20 Many studies have focused
on hydrophobically modified GC derivatives, such as GC-5-b-
cholanic acid, GC-deoxycholic acid, GC-N-acetyl histidine,
GC-cholesterol, GC-hydrotropic oligomer, GC-protoporphyrin,
GC-tocol, and GC-ergocalciferol succinate conjugates, because
of their amphiphilic structure.14,21–28 These polymeric
amphiphiles can form monodispersed self-aggregated nano-
particles in aqueous media and show good stable nanopar-
ticle structures in physiological conditions. Many
investigations have confirmed that these nanoparticles can
be used as carriers for hydrophobic medicines, gene delivery,
and medical diagnosis.29–36
Synthesis of Octahydrogenated Retinoic Acid
A suspension of retinoic acid and 10% palladium on carbon
in anhydrous cyclohexane/ethanol (1:1 v/v) was stirred in
reactor under H2 (4.04 3 105 Pa) for 24 h. The reaction mix-
ture was filtered through a pad of Celite. The filtrate was
concentrated under reduced pressure and the residue was
purified by silica gel column chromatography (petroleum
ether/ethyl acetate, 13:1 v/v) to afford OR as pale yellow oil.
1H NMR (600 MHz, CD3OD, d): 2.28 (dd, J 5 1.2, 6.0 Hz, 1H;
CH2), 2.25 (dd, J 5 1.2, 5.4 Hz, 1H; CH2), 2.10 (dt, J 5 1.8, 7.8
Hz, 1H; CH2), 2.07 (dt, J 5 1.8, 7.8 Hz, 1H; CH2), 1.93
(t, J 5 6.0 Hz, 2H; CH2), 1.60 (s, 3H; CH3). 13C NMR (150
MHz, CD3OD, d): 177.1 (C@O), 138.7 (C6), 127.4 (C5), 42.7
(C14), 41.1 (C2), 38.3 (C8), 38.1 (C9), 35.9 (C10), 35.2 (C1),
33.8 (C4), 31.4 (C13), 27.9 (C16), 27.9 (C17), 23.6 (C11),
23.6 (C7), 20.2 (C19), 20.1 (C3), 20.1 (C18), 20.1 (C20).
Preparation of OR-GC Nanoparticles
OR-GC conjugates were prepared by conjugation of GC with
OR in the presence of NHS and EDC, which produces amide
linkages between the amine groups of GC and carboxyl acid
groups of retinoic acid, as previously reported for other
amphiphilic polymers.37 GC was purified by filtration and
dialyzed against pure water for three times. The purified GC
(100 mg, 0.189 lmol) was dissolved in pure water (10 mL),
and OR (60 mg, 195 lmol) dissolved in methanol (10 mL)
was added under vigorous stirring. The chemical modifica-
tion was initiated by adding equal amounts (1.5 equiv/OR)
of NHS and EDC. Thereafter, the reaction was allowed to pro-
ceed for 24 h at room temperature. The solution was dia-
lyzed using dialysis tube (MW cutoff 5 12,000–14,000) for 3
days with 12 exchanges to remove unreacted chemicals. The
dialysis was performed by three steps [water/methanol (1:4
v/v), water/methanol (1:1 v/v), and pure water at days 1, 2,
and 3, respectively]. Finally, the dialyzed solution was lyophi-
lized for 2 days to produce a white, cotton wool-like product.
The chemical structure of OR-GC conjugates was analyzed
using 1H NMR spectra in CD3OD/D2O (3:1 v/v) at 600 MHz
(Bruker). FTIR spectra were recorded on Fourier-transform
infrared spectrometer (Nicolet 6700, Thermo Fisher Scien-
tific, USA) in KBr discs. The degree of substitution (DS)
defined as the number of OR per 100 sugar residues of GC
Few nanoparticle carriers for pesticides are available. In vitro
evaluation of GC-based nanoparticles for pesticide delivery
has yet to be reported. Therefore, we attempted to prepare
octahydrogenated retinoic acid (OR)-conjugated GC (OR-GC)
nanoparticles as a novel environment-friendly carrier that
can contain AZA in aqueous media. To produce AZA-loaded
OR-GC (AZA-OR-GC) nanoparticles, we encapsulated AZA into
the OR-GC nanoparticles by a simple dialysis method. To
evaluate the characteristics of AZA-OR-GC in vitro, its drug
release profile, cytotoxicity, and cell cycle analysis were stud-
ied in comparison with those of free AZA.
EXPERIMENTAL
Materials
GC (MW 5 5.3 3 105, degree of deacetylation 5 91%) was
purchased from Sigma-Aldrich Trading. All-trans retinoic acid
was purchased from Yuancheng Gongchuang Technology.
AZA (purity 45%, the experiments mentioned AZA weight
are the weight of the active ingredient) was produced by the
Key Laboratory of Pesticide and Chemical Biology of the Min-
istry of Education, South China Agricultural University, China.
1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochlor-
ide (EDC) and N-hydroxysuccinimide (NHS) were purchased
from Aladdin Chemistry. Methanol and anhydrous dimethyl
sulfoxide (DMSO) were purchased from Dikma Technologies.
Pure water used in all the experiments was obtained by
ELGA Pure Lab-ultra water purification system (resistance
1
was determined by H NMR.
Critical Micelle Concentration
The critical micelle concentration (CMC) of OR-GC was deter-
mined using the fluorescence probe method.9 Briefly, a
known amount of pyrene in tetrahydrofuran was added to
each of a series of 10-mL vials and the tetrahydrofuran was
evaporated at room temperature. The polymer concentration
varied from 1025 to 1 mg mL21, the final concentration of
pyrene was set to 6 3 1027 M for all samples, and then
sonicated for 30 min. The sample solutions were heated at
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filtered through a 0.8-lm membrane filter and then lyophi-
lized to give a pale yellow powder. The drug loading effi-
ciency of AZA-OR-GC nanoparticles was measured by high-
performance liquid chromatography (Agilent 1100 series).
The samples were analyzed for the AZA concentration by
reverse-phase HPLC system with Agilent TC C18 column (4.6
mm 3 250 mm) at a constant temperature of 30 ꢀC. The
mobile phase consisted of 65:35 (v/v) methanol/water and
was delivered at a flow rate of 1.0 mL min21. Eluted com-
pounds were detected at 218 nm using diode array detector.
Characterization of AZA-OR-GC Nanoparticles
The mean diameter and f-potential of OR-GC and AZA-OR-GC
nanoparticles were measured by dynamic light scattering
(DLS) using Malvern Zetasizer 90 (Malvern Instruments,
England). The sample concentration was kept at 1.667 mg
mL21 in pure water. The morphology of nanoparticles was
observed by transmission electron microscopy (TEM) (Tecnai
G2 Spirit, FEI, Netherlands), operated at an acceleration volt-
age of 120 kV. Each sample (2 mg mL21 in pure water) was
placed on a 300-mesh copper grid coated with carbon. Sub-
sequently, the sample was dried and negatively stained using
2% phosphotungstic acid dye solution.
In Vitro AZA Release Profile of AZA-OR-GC Nanoparticles
To determine the in vitro release profile of AZA from OR-GC
nanoparticles, lyophilized AZA-OR-GC nanoparticles (8 mg)
were dispersed in 2 mL of phosphate-buffered saline (PBS)
(pH 7.2). The resulting solution was placed into a cellulose
membrane dialysis tube (MW cutoff 5 12,000–14,000). The
dialysis tube was placed in 30 mL of PBS buffer and gently
shaken at 27 ꢀC in water bath at 100 rpm. At various time
points, the medium was refreshed. The concentration of AZA
was determined by UV–vis spectroscopy (Shimadzu, Japan).
FIGURE 1 Schematic representation of the synthetic route for
the glycol chitosan-octahydrogenated retinoic acid (OR-GC)
conjugates (A) and chemical structure of azadirachtin (B). Sche-
matic diagram of AZA-loaded OR-GC nanoparticles (C).
Cytotoxicity of AZA-OR-GC Nanoparticles
Spodoptera litura cultured ovarian cell lines Sl-1 obtained
from Sun Yat-sen University, College of Life Sciences
(Guangzhou, China) were routinely cultured in antibiotic-free
Grace’s medium containing 7% fetal bovine serum, 0.3%
yeast extract, and 0.3% lactalbumin hydrolysate. The cytotox-
icity of OR-GC, free AZA, and AZA-OR-GC nanoparticles was
evaluated by the MTT assay. Cells were seeded at a density
of 2 3 104 cells per well in 96-well flat-bottomed plates,
alloꢀwed to adhere for overnight, and incubated for 24 h at
27 C in 0.4% CO2. Five milligrams of AZA dissolved in 1 mL
of DMSO was prepared for AZA stock solution, followed by
sonication for 5 min. The stock solutions of AZA-OR-GC
nanoparticles in PBS (pH 7.2) were prepared by mixing 26.7
mg of OR-GC and 6.7 mg AZA by a dialysis method. Each
stock solution was diluted with culture medium to obtain a
concentration in the range of 0.5–50 lg mL21. DMSO (0.1%)
was used as the control and empty OR-GC also tested as
vehicle controls for the same concentrations. After 24, 48,
72, and 96 h of treatment, 10 lL MTT (5 mg mL21) was
added to each well and the cells were incubated further for
4 h at 27 ꢀC. The medium was removed and each well was
ꢀ
40 C for 3 h to equilibrate the pyrene and the nanoparticles
and left to cool overnight at room temperature. Fluorescence
spectra were measured at the excitation wavelength (kex) of
355 nm with a Hitachi F-4600 Fluorescence spectrophotome-
ter (Japan). Both excitation and emission bandwidth were
set at 2.5 nm, and the emission wavelength was 350–450
nm for emission spectra.
Preparation of AZA-Loaded OR-GC (AZA-OR-GC)
Nanoparticles
AZA-OR-GC nanoparticles were prepared by the dialysis
method. The synthesized OR-GC conjugates (100 mg) were
dissolved in 10 mL of water/methanol (1:1 v/v) and AZA
(25–100 mg) in 2 mL of methanol was added to the OR-GC
solution. The solution was vigorously stirred for 12 h at
room temperature and then dialyzed against pure water
using dialysis tube (MW cutoff 5 12,000–14,000). After dialy-
sis for 2 days, the solution was centrifuged at 10,000g for
30 min to remove the unloaded AZA. The supernatant was
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cells were incubated with OR-GC, AZA, and AZA-OR-GC at 27
ꢀC for serial times (24, 48, 72, and 96 h). After harvested,
cells were washed three times with 1 mL of PBS (pH 7.2)
and fixed in ice-cold 70% ethanol. After refrigerated over-
night at 4 ꢀC, the cells were washed with PBS to remove
residual ethanol and resꢀuspended in propidium iodide stain
buffer for 30 min at 27 C. The cell cycle phases of the total
cell population (G0/G1, S, and G2/M) were analyzed using
Becton Dickinson flow cytometer (USA).
RESULTS AND DISCUSSION
Preparation of OR-GC Nanoparticles
OR-GC is an amphiphilic polymer consisting of hydrophilic
and hydrophobic segments. In the aqueous phase, OR serves
as a hydrophobic core of polymers surrounded by hydro-
philic outer shells. Thus, the inner core can serve as a nano-
container for hydrophobic pesticides. As
a nanosized
pesticide carrier, biocompatible and biodegradable GC was
hydrophobically modified by OR in the presence of NHS and
EDC [Fig. 1(A)]. Formation of the amide linkage between GC
1
and OR was demonstrated by the FTIR and H NMR spectra.
FIGURE 2 FTIR spectra of GC (a) and OR-GC (b).
Figure 2 shows the FTIR spectra of GC and OR-GC. The
absorption peaks of GC are as follows: 3438 cm21 (tOAH
overlapped with tNAH), 2919 and 2873 cm21 (aliphatic
added 100 lL of DMSO. The absorbance was measured at
570 nm by a microplate reader (Bio-Rad, USA).38
t
CAH), 1664 cm21 (amide I band, tC@O of acetyl group), 1600
Cell Cycle Analysis by Flow Cytometry
cm21 (tNAH), 1461–1376 cm21 (tCAH), and 1064 cm21
(skeletal vibrations involving the tC@O). Compared with that
of GC, the spectrum of OR-GC showed an intense peak at
Cells were plated in plastic culture dishes and cultured at
27 ꢀC in 0.4% CO2. When the density was 106 cells mL21
,
FIGURE 3 1H NMR spectra of GC in D2O (concentration 5 25 mg mL21) (a), OR-GC in CD3OD/D2O (3:1 v/v) (concentration 5 20 mg
mL21) (b), and OR in CD3OD (concentration 5 20 mg mL21) (c).
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the aggregation behavior of surfactants or polymers in polar
solutions. When the concentration of OR-GC was increased,
the intensity ratio (I1/I3) decreased significantly with the
transfer of pyrene from an aqueous polar environment to a
more hydrophobic medium. The CMC of OR-GC was deter-
mined to be 13 lg mL21 (Fig. 4). These results suggest that
the micelle core was very hydrophobic.
Preparation of AZA-OR-GC Nanoparticles
AZA-OR-GC nanoparticles were simply prepared using a dial-
ysis method and the overall characteristics of the products
are summarized in Table 1. In aqueous environments, the
hydrophilic GC is situated outside of the molecules because
of hydrophilic interactions with water and the insoluble AZA
is found inside the molecules because of their hydrophobic
intermolecular and intramolecular interactions.22,28 To deter-
mine the loading capacity, we varied the feed weight ratio of
AZA from 25 to 100 wt % and found that the loading effi-
ciency of AZA into OR-GC nanoparticles decreased with
increasing amount of loaded AZA. When the AZA content in
OR-GC was 25 wt %, the maximum loading efficiency of
AZA-OR-GC nanoparticles was about 74%, which is attrib-
uted to the presence of hydrophobic inner cores. An impor-
tant observation is that the AZA-OR-GC nanoparticles were
well dispersed in aqueous medium. However, when the feed
amount of AZA was increased to above 100 wt %, the excess
AZA molecules precipitated during dialysis and the loading
efficiency significantly decreased to about 43%.
FIGURE 4 Plot of the intensity ratio I1/I3 (from pyrene excitation
spectra) as a function of log C.
1560 cm21 (amide II band, tNAH), an increased peak at
1733 cm21 assigned to the tC@O of amide bond in synthe-
sized OR-GC conjugate, and an increase in the amide I bend-
ing peak at 1650 cm21, which confirmed the amide linkage
between GC and OR.
1H NMR spectrum and results are shown in Figure 3. The
proton signals of GC are as follow: 1.98 ppm (CH3, methyl
protons of the acetyl group of N-acetamidoglucose units),
2.63 ppm (CH, Cb sugar protons of the N-unsubstituted glu-
cosamine units), 3.04–4.16 ppm (CH, Cc to Ch sugar protons),
and 4.38 ppm (CH, Ca anomeric sugar proton).14,28 Com-
pared with those of GC, the proton signals of OR-GC showed
peaks at 0.60–1.60 ppm, belonging to the high field signals
of OR. These results indicate that the OR group had been
incorporated into the GC. The DS, defined as the number of
OR molecules per 100 sugar residues of GC, was 20.4. On
average, OR-GC conjugates had 351 molecules of OR in one
GC polymer.
Based on the results of DLS analysis, the average diameter of
AZA-OR-GC increased with increasing loading content. These
observations show that AZA molecules are wrapped in the
hydrophobic inner cores of OR-GC and that entrapped AZA
molecules cause an increase in the mean diameter of the OR-
GC nanoparticles. The AZA-OR-GC nanoparticles spontane-
ously self-aggregated to spheroids with an average diameter
of 312–410 nm in aqueous conditions, as determined by DLS
and TEM (Fig. 5). With the aim of achieving higher loading
efficiency, we optimized the feed ratio of AZA into OR-GC to
25 wt %. Figure 5 displays the diameters of the OR-GC and
AZA-OR-GC (25 wt %) nanoparticles, which were almost
spherical and well-dispersed without aggregation.
Critical Micelle Concentration
The CMC of OR-GC was determined using the fluorescence
probe method. Pyrene molecules are reported to preferably
localize inside or close to the hydrophobic microenvironment
of micelles, varying with different molecular photophysical
properties.14 The emission intensity ratio of the first
(372 nm, I1) and third (382 nm, I3) peaks is used to monitor
In Vitro Release Profile of AZA-OR-GC Nanoparticles
To improve the efficacy of AZA, sustained release of AZA
from polymeric nanoparticles was studied. To evaluate the
potential of OR-GC nanoparticles as a pesticide carrier of
TABLE 1 Physical Characteristics of AZA-Loaded OR-GC Nanoparticles
Sample
Loading Content of AZA (wt %)
Loading Efficiency (%)
Diameter (nm)
f-Potential (mV)
OR-GC
–
–
257 6 12
312 6 16
379 6 18
410 6 10
38.1 6 0.8
28.3 6 0.5
21.2 6 0.3
25.3 6 0.3
AZA-OR-GC (25 wt %)
AZA-OR-GC (50 wt %)
AZA-OR-GC (100 wt %)
18.4 6 0.5
31.1 6 0.7
42.7 6 2.4
73.6 6 1.9
62.2 6 1.3
42.7 6 2.4
Mean diameter in pure water as measured by dynamic light scattering (n 5 3). The OR-GC and AZA-OR-GC nanoparticles concentrations are 1.667
mg mL21
.
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FIGURE 5 Particle size and morphological shapes of OR-GC (A)
and AZA-OR-GC nanoparticles (B). Average size of OR-GC and
AZA-OR-GC (25 wt %) nanoparticles (1.667 mg mL21 in pure
water) was measured using dynamic light scattering. Inset
image indicated TEM image of OR-GC and AZA-OR-GC (25 wt %)
nanoparticles in pure water (2 mg mL21).
FIGURE 7 Cytotoxicity of AZA-OR-GC (25 wt %) nanoparticles.
Inhibition of cell proliferation of each sample was measured
using MTT assay after treated for 48 h (A), 72 h (B), and 96 h
(C). Data represent mean 6 SD (n 5 3).
AZA, the release behavior of AZA from OR-GC nanoparticles
was assessed at 27 ꢀC in PBS (pH 7.2). During this test, the
sink conditions were maintained by regularly replacing the
dialysis medium. Figure 6 shows the in vitro AZA release
profile from AZA-OR-GC (25 wt %) nanoparticles. An initial
rapid release phase burst effect was observed within 12 h,
and 30% of the AZA was released from the nanoparticles.
After the first 12 h, the cumulative release of AZA gradually
FIGURE 6 AZA release profile from nanoparticles at 27 ꢀC in
PBS (pH 5 7.2). Data represent mean 6 SD (n 5 3).
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FIGURE 8 Cell cycle distribution of Sl-1 cells. Cells were incubated in OR, AZA, and AZA-OR-GC (25 wt %) for 24, 48, 72, and 96 h,
and analyzed by flow cytometry. The equivalent AZA concentration was 10 lg mL21
.
plateaued and release was sustained for the following 6
days. Approximately 65% of the AZA was released over 7
days, indicating the potential application of the nanoparticles
in sustained pesticide delivery systems. These two phases
may be controlled by a diffusion mechanism resulting from
partitioning between polymer nanoparticles and the sur-
rounding aqueous phase. The initial rapid release phase may
be due to weak interactions between the released AZA mole-
cules and the hydrophobic moiety of nanoparticles, and the
subsequent sustained release phase may be due to strong
interactions between the released AZA molecules and the
hydrophobic nanoparticles cores.28
AZA dissolved in DMSO and AZA-OR-GC (25 wt %) nanopar-
ticles in PBS was evaluated by determining their inhibitory
effects on Sl-1 ovarian cell proliferation (Fig. 6). Based on
the MTT test, empty OR-GC nanoparticles as a vector control
showed slight cytotoxicity varying from 0.5 to 50 lg mL21
.
By contrast, all of the samples exhibited significant cytotoxic-
ity against the Sl-1 cells, which was further enhanced by
increasing the time of exposure and AZA concentration.
When the Sl-1 cells were treated with 50 lg mL21 for 48 h,
inhibition of cell proliferation by AZA-OR-GC (25 wt %) was
53% compared with that by free AZA (65%). After 96 h of
treatment, inhibition of cell proliferation of AZA-OR-GC (25
wt %) nanoparticles increased to 77% compared with that
by free AZA (87%) at 50 lg mL21. These results show that
the cytotoxicity of AZA-OR-GC (25 wt %) against Sl-1 cells is
relatively lower than that of DMSO-based AZA, which may be
attributed to the slow and sustained release of AZA
Cytotoxicity of AZA-OR-GC Nanoparticles in Sl-1 Cells
Pesticide delivery carriers must be biocompatible and
environment-friendly for their successful utilization in the
field of agricultural chemicals. Thus, the cytotoxicity of free
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TABLE 2 Cell Cycle Distribution (20,000 Cells Per Count)
controlled release, achieving roughly a 65% of loaded drug in
7 days in PBS (pH 7.2). Compared with free AZA in organic
solvents, the AZA-OR-GC nanoparticles showed favorable inhi-
bition of cell proliferation and sustained drug release per-
formance in Sl-1 cells in vitro. Finally, environment-friendly
and controlled release AZA-encapsulated OR-GC formulations
showed great potential to apply in IPM.
Cell Cycle Distribution (%)
Treatment
Control
Apoptosis
G0/G1
S
G2/M
24 h
48 h
72 h
96 h
0.0
0.0
0.7
1.2
0.0
0.0
1.5
1.1
0.0
0.0
2.0
3.0
0.1
0.1
6.2
6.1
20.0
15.2
0.1
48.1
41.3
29.3
17.0
48.1
38.7
10.5
12.1
49.6
45.2
5.6
32.0
43.6
70.6
80.1
34.6
48.2
89.5
87.9
33.2
43.4
92.7
65.6
26.2
38.7
72.0
65.8
OR-GC
AZA
AZA-OR-GC
Control
OR-GC
2.9
ACKNOWLEDGMENTS
17.3
13.1
0.0
This research was financially supported by Agro-scientific
Research in the Public Interest of China (No. 200903052) and
Science and Technology Planning Project of Guangdong
Province, China (No. 2012B020309004). The authors thank
Zhuo-Qiang Zhou (College of Science, South China Agricultural
University) for the synthesis of OR, and Zhi-Wei Lei for NMR
spectrum analysis.
AZA
AZA-OR-GC
Control
OR-GC
0.0
17.2
10.8
1.7
AZA
AZA-OR-GC
Control
OR-GC
0.0
34.4
59.6
50.5
28.0
34.1
14.2
10.8
0.1
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