Nano-film pesticide for Schistosoma japonicum cercariae: Synthesis, characterization, toxicity and insecticidal effect

Article abstract of DOI:10.1039/c3ra44328k

To improve the efficiency of pesticides, two novel niclosamide derivatives have been synthesized by adding polyethylene glycol groups (PEG) with different lengths to the niclosamide core. The derivatives were characterized by infrared spectroscopy (IR) an

Full text of DOI:10.1039/c3ra44328k

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Nano-film pesticide for Schistosoma japonicum
cercariae: synthesis, characterization, toxicity and
Cite this: RSC Adv., 2013, 3, 19956
insecticidal effect†
Yibao Li,a Yunzhi Xie,a Chunhua Liu,a Wei Guo,a Xiaokang Li,a Xun Li,a
Qingdao Zeng and Xiaolin Fan
Received 13th August 2013
Accepted 2nd September 2013
b
a
*
*
DOI: 10.1039/c3ra44328k
www.rsc.org/advances
To improve the efficiency of pesticides, two novel niclosamide water solubility,5,6 so anti-cercarial effect of niclosamide is not
derivatives have been synthesized by adding polyethylene glycol good in actual application.
groups (PEG) with different lengths to the niclosamide core. The
To improve the efficiency of pesticides, nano-lm pesticide
derivatives were characterized by infrared spectroscopy (IR) and (derivatives of niclosamide) would be expected oating on the
magnetic resonance (1H NMR). The results from brewster angle air–water interface. The development of nanomedicine might
microscopy (BAM) revealed that these two derivatives formed a self- open up potential novel applications in agriculture and
diffusion thin film at the air–water interface. In addition, Langmuir– biotechnology,7–9 especially in biomedical and pharmacology.10
Blodgett (LB) films on solid substrate have been used to investigate Besides it had low pollution to environment. Shi et al. had
the formation mechanism of the thin film at the air–water interface, achieved nanomedicine for targeted drug delivery.11 Zhang et al.
combined with atomic force microscopy (AFM). It was suggested that had used nanospheres to prepare slow-release nanomedicine.12
the height of the monolayer at the air–water interface is consistent New opportunity and wide application prospect for the research
with the theoretical length of niclosamide derivatives. Further on pesticide were provided by nanomedicine.
experimental results indicated that these two compounds exhibit
Due to the non-toxic and good biocompatibility, PEG has
relatively low cytotoxicity to HeLa cells, but have higher efficiency to been approved by the FDA as intravitreal injection of medicinal
anti-cercariae compared with that of pristine pesticide.
polymer. The PEG polymers have many advantages: (1) solu-
bility of the drug is increased, (2) half life enzymatic degrada-
tion is decreased, immunogenicity and period is prolonged, (3)
maximum plasma concentration is lowered, the uctuation of
blood concentration is attened, (4) antigenicity is reduced,
toxicity is also reduced.13–18 Shen et al. had used PEG to improve
solubility of the drug.19 Robinson et al. had used PEG to extend
the half-life of nanomaterials in vivo.20 In addition, Watkins
et al. had used PEG to synthesise amphiphilic molecules which
can self-diffuse at the water–air interface.21
Herein, this paper focuses on synthesis of novel niclosamide
derivatives with PEG that can self-diffuse at the water–air
interface, which will rapidly kill S. japonicum cercariae. Novel
derivatives of niclosamide (Scheme 1, 2a and 2b) have been
synthesized by coupling PEG with the niclosamide core. The
lm-forming ability of the novel niclosamide derivatives at the
1. Introduction
The cercarial stage of the schistosome life-cycle is the only
infectious stage. The cercarial stage is also the most fragile stage
in the life cycle of schistosome.1 More than 98% of cercariae
oat on the water surface, so it is possible to effectively control
the risk of cercariae infection by the use of a thin cercaricide
lm oated on the water.2 Niclosamide (see Scheme 1,
compound 1) has a long track-record as a killer of the tapeworm
and snails that harbor schistosome.3 Moreover, niclosamide
can effectively prevent Schistosoma japonicum cercariae from
entering a human host by rendering cercariae uninfective with
very short contact time.4 Unfortunately, niclosamide has low
aKey Laboratory of Organo-Pharmaceutical Chemistry, Gannan Normal University,
Ganzhou 341000, P. R. China. E-mail: Fanxl2013@gnnu.cn; Fax: +86 (0)797 8393536
bNational Center for Nanoscience and Technology (NCNST), Beijing 100190, P. R.
China. E-mail: zengqd@nanoctr.cn; Fax: +86 (0)10 82545548
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c3ra44328k
Scheme 1 The synthesis of 2 (2a and 2b).
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water–air interface was observed by the BAM. To further speed of 7.5 cm2 minꢂ1 aer waiting 20 min for the evapo-
study the formation mechanism of lm, LB technique has ration of the solvent.
been used at the air–water interface, combined with AFM.
In addition, the cell toxicity and anti-cercarial ability were
further investigated.
2.4 AFM images of nano-lm pesticides
The freshly cleaved mica plate was rst immersed into the pure
water. The compounds 2a and 2b in chloroform solutions
were then spread on the sub-phase, Aer the spread lm was
2. Experimental section
2.1 Synthesis
compressed to a constant surface pressure (5, 10, 20 mN mꢂ1
)
and waiting for 5 min for the equilibrium of the monolayer, the
mica substrates were vertically lied through the monolayer at
a speed of 1 mm minꢂ1. Only one layer was transferred onto
the mica surface, allowing the water to evaporate in air at room
temperature. The mica was covered with watch glasses to
protect their surfaces from dust. This may also help create a
relatively conned space and make the evaporation of solvents
slower as compared to that in an open-air environment. AFM
measurements were performed by using a Nanoscope IIIa
(Veeco Metrology, USA).
2.1.1 2-[2-(2-Hydroxyethoxy)ethoxy]ethoxyl
niclosamide
(2a). 2a was synthesized according to the literature proce-
dure.22,23 Niclosamide (23.0 g, 70 mmol) was dissolved in 1,4-
dioxane (100 mL) at 353.15 K in a 250 mL three-neck ask.
K2CO3 (9.2 g, 70 mmol) and Bu4NBr (2.30 g, 7 mmol) were added
with stirring. 2-(2-(2-Hydroxyethoxy)ethoxy)ethyl 4-methyl-
benzenesulfonate (12.77 g, 42 mmol) dissolved in l,4-dioxane
(30 mL) was added dropwise and the ensuing mixture was
heated under reux for 12 h, and allowed to cool. Water (200
mL) was then added and the mixture was shaken in a separating
funnel. The organic phase was separated, and the water phase
extracted with CH2Cl2 (50 ꢀ 3 mL). The combined organic layer
was dried over anhydrous Na2SO4. Aer removing the solvent
under reduced pressure, the crude product was puried by
column chromatography, and a slightly yellow solid 2a was
2.5 Toxicity test
The cell survival rates of 2a and 2b were detected by using MTT
assay. HeLa cells were plated on 96 well tissue culture plate in
an atmosphere of 5% CO2, 95% air at 37 ꢁC to adhere for 12 h.
Mixed the cell pellet with 2a solution (100 mL per well), the nal
concentrations of 10, 20, 30, 40, 50, 60, 70, and 80 mM, the same
procedure for 2b and compound 1, as experimental group.
Mixed the cell pellet with RPMI 1640 medium containing 0.2%
DMSO (100 mL per well), as reference group. Aer cell culture
incubator for 24 h, added 20 mL of MTT/PBS (5 mg mLꢂ1) to each
well and cell culture incubator for 3 to 4 h. Remove culture
medium, added 100 mL of DMSO to each well and measured the
absorbance in each well, including the blanks, at 570 nm in a
microtiter plate reader. The reference wavelength was 690 nm.
The detail mathematical description of the cell survival rate is
given by
ꢁ
obtained. Yield 79%. mp 84–86 C IR (KBr) n 3443, 3302(N–H,
O–H), 3077, 2893(CH2), 1673, 1592, 1542, 1509(Ar), 1402,
1340(N]O), 1271, 1207, 1118(C–O–C), 1048(Ar–Cl), 891, 827,
743(Ar), 675 cmꢂ1; dH (400 MHz, CDCl3) 10.62 (1H, s), 8.88 (1H,
d, J 9.2), 8.35 (1H, d, J 2.6), 8.26 (1H, d, J 2.8), 8.23 (1H, dd, J 9.3,
2.6), 7.50 (1H, dd, J 8.9, 2.8), 7.11 (1H, d, J 8.9), 4.49 (2H, t, J 4.8),
3.96 (2H, t, J 4.8), 3.69–3.64 (4H, m), 3.60–3.57 (2H, m), 3.52
(2H, t, J 4.8). HR-MS: calcd for C19H20Cl2N2O7: 459, [M + H]+.
Found: 459.
2.1.2 2-{2-[2-(2-Hydroxyethoxy)ethoxy]ethoxy}ethoxyl niclo-
samide (2b). The synthesis and separation methods for 2b were
similar to those adopted for 2a, and a slight yellow liquid was
obtained. Yield 70%. dH (400 MHz, CDCl3) 10.58 (1H, s), 8.79
(1H, d, J 9.2), 8.27 (1H, dd, J 2.8), 8.17–8.11 (2H, m), 7.43 (1H, dd,
J 8.9, 2.8), 7.08 (1H, d, J 8.9), 4.45 (2H, t, J 4.8), 3.92 (2H, t, J 4.8),
3.66 (2H, t, J 4.8), 3.64–3.61 (2H, m), 3.58–3.51 (8H, m), 2.79 (1H,
s). MS: [M + H]+ m/z (%) 503.3.
cell survival rate (%) ¼ (absorbance of experimental
group/absorbance of reference group) ꢀ 100%.
All experiments were performed at least in triplicate. The
ANOVA test was used to examine the statistical signicance of
differences. A p-value less than 0.05 was considered to indicate a
statistically signicant difference.
2.2 BAM images of nano-lm pesticides
Exploring the morphology of the lm, the compounds 2a and 2b
was investigated by this method in the following experiments.
Compounds 2a and 2b was spread on the pure water surface
with different concentration in chloroform. The lm structure
of the molecular was investigated by the BAM JB04 (POWER-
EACH, Shanghai). The water was obtained from a pure water
system.
2.6 Anti-cercarial activity experiments
Compound 2b was dissolved in dimethyl sulphoxide (DMSO),
and diluted into concentrations with distilled water as follows:
0.6 mM, 1.3 mM, 2.5 mM, 5.0 mM and 10.0 mM. S. japonicum
cercariaes were collected from the infected O. hupensis. The O.
hupensis were induced to shed cercariae by exposing them to
bright light for 2 h, and cercariae were then transferred by metal
2.3 Surface pressure–area (p–A) isotherms
The monolayers were formed by spreading a chloroform spatula from the water surface to a plate containing a dilute
solution (1 ꢀ 10ꢂ3 mol Lꢂ1) onto the surface of pure water. solution of compound 2b, the activity of the cercariae was
Surface pressure–area (p–A) isotherms were recorded on a explored by biological microscopy at 30 min, 60 min, 90 min
KSV (KSV 1100, Finland) instrument with a compressing and 120 min. The same procedure for 2a.
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structure appeared on the water surface while the concentration
of compound 2b is 7.5 ꢀ 10ꢂ6 mmol cmꢂ2 (Fig. 2b). However,
white concentric structure cannot be found while the concen-
tration of compound 2a is 7.5 ꢀ 10ꢂ6 mmol cmꢂ2 (Fig. 1b). In
addition, white concentric structure of compound 2b (Fig. 2c
and d) is denser and more uniform than 2a (Fig. 1c and d) at the
same concentration. Concentric structure and banded structure
(Fig. 2e and f) also appeared on the water surface at 4.5 ꢀ 10ꢂ5
mmol cmꢂ2, but banded structures cannot be found at this
concentration for compound 2a (Fig. 1e). The results from BAM
revealed that these two derivatives could form self-diffusion
thin lm at the air–water interface, and the formation ability for
lms of compound 2b is better than that of compound 2a.
Fig. 1 BAM images the films of compound 2a with different concentrations at
the air–water interface. The morphologies and relative concentrations: (a) black;
(b) 7.5 ꢀ 10ꢂ6 mmol cmꢂ2; (c) 1.5 ꢀ 10ꢂ5 mmol cmꢂ2; (d) 3 ꢀ 10ꢂ5 mmol cmꢂ2; (e)
3.2 AFM images of nano-lm pesticides
4.5 ꢀ 10ꢂ5 mmol cmꢂ2; (f) 7.5 ꢀ 10ꢂ5 mmol cmꢂ2
.
These derivatives formed self-diffusion thin lm at the air–
water interface, but the mechanism of lm-forming is not clear.
In order to investigate the detailed microcosmic aggregation
structures, LB technique has been used at the air–water inter-
face, combined with AFM. Fig. 3 shows the p–A isotherms for
the monolayer lms of compounds 2a and 2b at the air–water
interface at 20 ꢁC. The experimental results show two possesses
properties of self-diffusion and surface lm formation at the
air–water interface. When surface pressure less than 35(48) mN
mꢂ1, compound 2a (2b) could form stable monolayers on the
water surface with a limiting molecular area of around 1.1–2.5
(1.6–6.5) nm2 per molecule, depending on the spacer length, as
shown in Fig. 3.
The LB lm deposited from the water surface is very at
(Fig. 4 and 5). The AFM height images in Fig. 4 showed planar
structures deposited on mica surface from monolayer lms of
compound 2a with 0.70 nm, 0.99 nm and 1.50 nm, respectively.
The AFM height images in Fig. 5 showed planar structures
deposited on mica surface from monolayer lms of compound
2b with 0.85 nm, 1.07 nm and 1.75 nm, respectively. With the
increase of surface pressure, the height of the lm is larger,
which may be attributed to the angle between the niclosamide
derivatives and water. Theoretical calculations show that
3. Results and discussions
3.1 BAM images of nano-lm pesticides
To observe the morphologies of two novel niclosamide deriva-
tives (compounds 2a and 2b) at the air–water interface, BAM
images were recorded under different concentration. The
concentrations are 0, 7.5 ꢀ 10ꢂ6, 1.5 ꢀ 10ꢂ5, 3 ꢀ 10ꢂ5, 4.5 ꢀ
10ꢂ5 and 7.5 ꢀ 10ꢂ5 mmol cmꢂ2, respectively. Compound 2a and
2b could form apparent molecular lms at the air–water inter-
face. For compound 2a, the lm cannot be found at low
concentration (Fig. 1a and b). With the concentration increasing,
white concentric structure appeared while the concentration of
compound 2a is 1.5 ꢀ 10ꢂ5 mmol cmꢂ2 (Fig. 1c). It shows that
lm has emerged. With the further adding of compound 2a, the
concentric structure become denser and more uniform (Fig. 1d
and e). It is interesting that concentric structure and banded
structure are observed while the concentration of compound 2a
is 7.5 ꢀ 10ꢂ5 mmol cmꢂ2 (Fig. 1f). It shows that lm has stacked.
The lm becomes thicker.
Compared Fig. 1 with 2, the concentration of formation lm
for compound 2b is lower than that of 2a. White concentric
Fig. 2 BAM images the films of compound 2b with different concentrations at
the air–water interface. The morphologies and relative concentrations: (a) black;
(b) 7.5 ꢀ 10ꢂ6 mmol cmꢂ2; (c) 1.5 ꢀ 10ꢂ5 mmol cmꢂ2; (d) 3 ꢀ 10ꢂ5 mmol cmꢂ2; (e)
Fig. 3 p–A isotherms for the monolayer films of compounds 2a and 2b at the
air–water interface at 20 ꢁC.
4.5 ꢀ 10ꢂ5 mmol cmꢂ2; (f) 7.5 ꢀ 10ꢂ5 mmol cmꢂ2
.
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Fig. 6 Cell viability (%) estimated by MTT proliferation test versus incubation
concentrations of compound 2a and 2b, HeLa cells were cultured in the presence
of 0–80 mmol Lꢂ1 compound 2a and 2b at 37 ꢁC for 24 h. Results are shown as
mean ꢃ S.D. of six independent samples (*: p < 0.05; **: p < 0.01, ***: p < 0.001).
Table 1 The mortality rate of Schistosoma japonicum cercariae for the cercar-
icide 2aa (25 ꢁC)
Mortality (%)
Concentration
(mM)
30 min
60 min
90 min
120 min
Fig. 4 AFM height images and molecular models of compound 2a one-layer LB
10.0
5.0
2.5
1.3
0.6
100.0
76.0 ꢃ 1.0
59.8 ꢃ 3.8
0.0
0.0
0.0
100.0
100.0
98.6 ꢃ 2.9
82.2 ꢃ 3.0
61.1 ꢃ 3.4
0.0
100.0
100.0
100.0
93.2 ꢃ 2.7
71.4 ꢃ 2.2
0.0
100.0
100.0
100.0
98.7 ꢃ 1.3
84.4 ꢃ 0.4
0.0
films at different surface pressure. (a) 5 mN mꢂ1, (b) 10 mN mꢂ1, (c) 20 mN mꢂ1
,
respectively.
Blank
a
All experiments were performed at least in triplicate.
Table 2 The mortality rate of Schistosoma japonicum cercariae for the cercar-
icide 2ba (25 ꢁC)
Mortality (%)
Concentration
(mM)
30 min
60 min
90 min
120 min
10.0
5.0
2.5
1.3
0.6
100.0
100.0
100.0
100.0
90.4 ꢃ 2.4
81.0 ꢃ 2.9
0.0
100.0
100.0
100.0
95.9 ꢃ 0.1
86.5 ꢃ 1.9
0.0
100.0
100.0
100.0
100.0
90.6 ꢃ 2.1
0.0
85.2 ꢃ 1.2
74.6 ꢃ 0.7
2.8 ꢃ 1.4
0.0
Blank
0.0
a
All experiments were performed at least in triplicate.
Fig. 5 AFM height images and molecular models of compound 2b one-layer LB
films at different surface pressure. (a) 5 mN mꢂ1, (b) 10 mN mꢂ1, (c) 20 mN mꢂ1
,
2b. Firstly, HeLa cells incubated with 2a and 2b for 24 hours,
cell viabilities are more than 89% at 20 mM (Fig. 6). With
increasing the concentration, the cell survival rate is more than
80% in 50 mM compound 2a and compound 2b (Fig. 6).
respectively.
niclosamide derivatives nearly lay at at a surface pressure of 5
mN mꢂ1. With the increase of surface pressure, niclosamide
derivatives gradually stand. Niclosamide derivatives nearly
totally stand at surface pressure of 20 mN mꢂ1. Under the same
surface pressure, the height of compound 2a lm is bigger than
compound 2b. This is due to the hydrophilic group of
compound 2a is longer than compound 2b.
3.4 Anti-cercarial activity experiments
In order to study anti-cercarial ability of nano-lm pesticide, the
anti-cercarial activity experiments is performed in different
concentration of compound 2a and 2b. The mortality rate of S.
japonicum cercariae for compound 2a and 2b was high, not only
at a high concentration of 10.0 mM but also at a low concen-
tration of 0.6 mM. Firstly, cercarial incubated with 2a and 2b for
30 min, mortality of cercariae are more than 100% at 10.0 mM
3.3 Cytotoxicity test
In order to study niclosamide derivatives' cytotoxicity, the MTT (Tables 1 and 2). With reducing the concentration, the mortality
assay was used to detect cell survival rate of compound 2a and of cercariae is more than 84% in 0.6 mM compound 2a and
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compound 2b (Tables 1 and 2). That implies the cercaricide 2a
and 2b maintained excellent bioactivity against cercariae in
spite of the hydroxyl group of niclosamide being replaced by the
PEG linker.
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4. Conclusion
The niclosamide derivatives have been synthesized and char-
acterized. The BAM experimental results show the niclosamide
derivatives can form self-diffusion lm at the air–water inter-
face. In order to investigate the detailed microcosmic aggrega-
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19960 | RSC Adv., 2013, 3, 19956–19960
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