Journal of Biotechnology 160 (2012) 97104

Contents lists available at SciVerse ScienceDirect

Journal of Biotechnology
journal homepage: www.elsevier.com/locate/jbiotec

Chitosan and its quaternized derivative as effective long dsRNA carriers

targeting shrimp virus in Spodoptera frugiperda 9 cells
Gatesara Theerawanitchpan a,b , Nattika Saengkrit c , Warayuth Sajomsang c , Pattarapond Gonil c ,
Uracha Ruktanonchai c , Somsak Saesoo c , Timothy W. Flegel a,b,d , Vanvimon Saksmerprome a,d,
a

Center of Excellence for Shrimp Molecular Biology and Biotechnology (Centex Shrimp), Bangkok, Thailand
Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok, Thailand
c
National Nanotechnology Center (NANOTEC), National Science and Technology Development Agency (NSTDA), Thailand Science Park, Pathumthani, Thailand
d
National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Thailand Science Park, Pathumthani, Thailand
b

a r t i c l e

i n f o

Article history:
Received 15 March 2012
Received in revised form 20 April 2012
Accepted 23 April 2012
Available online 30 April 2012
Keywords:
Yellow head virus (YHV)
RNA interference (RNAi)
Chitosan
Quaternized chitosan
Non-viral vectors
Spodoptera frugiperda

a b s t r a c t
RNA interference (RNAi) is a promising strategy to combat shrimp viral pathogens at lab-scale experiments. Development of effective orally delivered agents for double-stranded (ds)RNA is necessary for
RNAi application at farm level. Since continuous shrimp cell lines have not been established, we are developing a dsRNA-delivery system in Spodoptera frugiperda (Sf9) cells for studying in vitro RNAi-mediated
gene silencing of shrimp virus. Sf9 cells challenged with yellow head virus (YHV) were used for validating
nanoparticles as effective dsRNA carriers. Inexpensive and biodegradable polymers, chitosan and its quarternized derivative (QCH4), were formulated with long dsRNA (>100 bp) targeting YHV. Their morphology
and physicochemical properties were examined. When treated with chitosan and QCH4dsRNA complexes, at least 50% reduction in YHV infection in Sf9 cells relative to the untreated control was evident at
24 h post infection with low cytoxicity. Inhibitory effects of chitosan and QCH4dsRNA complexes were
comparable to that of dsRNA formulated with Cellfectin , a commercial lipid-based transfection reagent.
The natural and quaternized chitosan prepared in this study can be used for shrimp virus-specic dsRNA
delivery in insect cultures, and have potential for future development of dsRNA carriers in shrimp feed.
2012 Elsevier B.V. All rights reserved.

1. Introduction
Chitosan and its derivatives as non-viral vectors have gained
tremendous interest for their applications, especially for gene
transfer and RNAi delivery systems. Chitosan can encapsulate
nucleic acids, both DNA and RNA, and form nanopolyplexes via
ionic interactions. The nanopolyplexes are low toxicity, biodegradable and biocompatibility (De Smedt et al., 2000; Koping-Hoggard
et al., 2001). Chemical modications and the ratio of positively
charged chitosan and negatively charged nucleic acids are introduced to optimize stability of the nanopolyplex. Several previous
studies demonstrated the capacity of chitosan or chitosan derivative to carry and deliver plasmid DNA and RNA both in vivo
and in vitro systems (Koping-Hoggard et al., 2001; Rojanarata
et al., 2008; Techaarpornkul et al., 2010; Weecharangsan et al.,
2008). However, applications of chitosan are still limited due to
its insolubility in neutral and basic pH. Moreover, its low specicity and transfection efciency of chitosan should be overcome

Corresponding author at: Centex Shrimp, Faculty of Science, Mahidol University,

Bangkok 10400, Thailand. Tel.: +662 201 5870; fax: +662 354 7344.
E-mail address: vsaksmer@gmail.com (V. Saksmerprome).
0168-1656/$ see front matter 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jbiotec.2012.04.011

for its use in clinical trials (Kim et al., 2007). Quaternized chitosan
(QCH) was developed to improve solubility of natural chitosan at
pH 7. It was prepared by using commercially available Quat 188
under basic condition (Sajomsang et al., 2009a,b). The Quat 188
is an aqueous solution of N-(3-chloro-2-hydroxypropyl) trimethylammonium chloride (Xiao et al., 2012). It is well-known as a
quaternizing agent that introduce quaternary ammonium moiety
into the polymer backbone such as the one in starch (Heinze et al.,
2004), cellulose (Hashem et al., 2003), and chitosan (Sajomsang
et al., 2009a,b).
RNAi-based preventive approaches have shown promise against
shrimp viruses at lab-scale experiments. To date, intramuscularly
injection of dsRNA appears to be the most effective delivery method
for RNAi-mediated shrimp viral inhibition (Ongvarrasopone et al.,
2008; Saksmerprome et al., 2009; Tirasophon et al., 2007). There
remains a need to improve oral delivery method to make RNAimediated antiviral strategy feasible for shrimp farming. Due to the
lack of availability of continuous shrimp cell line, mosquito and
Spodoptera frugiperda 9 (Sf9) cell cultures have gained interest for
those who study shrimp-virus responses in molecular detail. They
have been reported to be immunopositive to white spot syndrome
virus (WSSV) and yellow head virus (YHV), suggesting likelihood of
shrimp viruses persistently replicating in insect cells (Sriton et al.,

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G. Theerawanitchpan et al. / Journal of Biotechnology 160 (2012) 97104

2009; Gangnonngiw et al., 2010). The insect cells could be useful not
only for studying RNAi-mediated gene silencing of shrimp virus but
also for validating dsRNA carriers with promising inhibitory effects
prior to testing in shrimp.
In this study, chitosan and its quarternized derivatives are prepared as previously described (Sajomsang et al., 2009a,b), and
formulated with bacterially expressed dsRNA (>100 bp) targeting
YHV RNA-dependent RNA polymerase (RdRp) gene. Complex formations were analyzed by physicochemical methods. The aim of
this study is to evaluate transfection efcacy and cell toxicity of the
chitosanviral specic dsRNA complexes in an insect cell system.
The effective chitosan nanoparticles determined from this study
would have further applications in development of shrimp feed
containing viral specic dsRNA.
2. Materials and methods
2.1. Preparation of chitosan and QCH4
The chitosan, with average molecular weights (Mw ) of 276 was
purchased from Seafresh Chitosan (lab) Co., Ltd. in Thailand. The
degree of deacetylation (DDA) of chitosan was determined to be
94% by 1 H NMR spectroscopy (Lavertu et al., 2003). N-(3-Chloro2-hydroxypropyl) trimethylammonium chloride (Quat 188) was
obtained from the Dow Chemical Company in Thailand. A dialysis
tubing with Mw cut-off of 12,00014,000 g/mol from Cellu Sep T4,
Membrane Filtration Products, Inc., (Segiun, TX, USA) were used
to purify chitosan derivative. Chitosan was dispersed in 1% (w/v)
acetic acid to prepare the stock solution at a nal concentration of
1 g/l. QCH4 was synthesized by quaternizing chitosan with N-(3chloro-2-hydroxypropyl) trimethylammonium chloride (Quat 188)
(Sajomsang et al., 2009a,b).
2.2. Characterization of chitosan and QCH4
All attenuated total reectance Fourier transform infrared (ATRFTIR) spectra were collected with a Nicolet 6700 spectrometer
(Thermo Company, USA) using the single-bounce ATR-FTIR spectroscopy (Smart Orbit accessory) with a diamond internal reection
element (IRE) at the ambient temperature (25 C). These spectra were collected by using rapid-scan software in OMNIC 7.0
with 32 scans and a resolution of 4 cm1 . The 1 H NMR spectra
were measured on AVANCE AV 500 MHz spectrometer (Bruker,
Switzerland). All measurements were performed at 300 K, using
the pulse accumulation of 64 scans and LB parameter of 0.30 Hz.
D2 O/CD3 COOD and D2 O were used as the solvents for dissolving 5 mg of chitosan and QCH4, respectively. The weight average
molecular weight (Mw ), number average molecular weight (Mn ),
and Mw /Mn of chitosan and QCH4 were determined by using the
gel permeation chromatography (GPC). It consists of Waters 600E
Series generic pump, injector, ultrahydrogel linear columns (Mw
resolving range 120,000 kDa), guard column, pollulans as standard
(Mw 5.9788 kDa), and refractive index detector (RI). All samples
were dissolved in acetate buffer pH 4 and then ltered through
VertiPure nylon syringes lters 0.45 m (Vertical chromatography
Co., Ltd., Thailand). The mobile phases, 0.5 M AcOH and 0.5 M AcONa
(acetate buffer pH 4), were used at a ow rate of 0.6 ml/min at 30 C.
Then the injection volume 20 l was used.

QCH4 solution were mixed with 1 g of dsRNA solution. The complexes were formed through a self-assembly mechanism after
pipetting, and subsequently incubated at room temperature for
15 min before use. Different weight ratios of chitosandsRNA and
QCH4dsRNA complex were prepared to investigate dsRNA binding afnity by gel retardation. Physicochemical characterization
and morphological analysis of the obtained nanoplexes were then
examined. Hydrodynamic diameter, polydispersity index (PDI) and
zeta potential of nanopolyplexes were determined by the Photon Correlation Spectroscopy machine and electrophoretic mobility
titration (NanoZS4700 nanoseries, Malvern Instruments, UK). The
samples were obtained as the average of three measurements
at 25 C. For morphology investigation, 5 l of each of dsRNA,
chitosan, QCH4, chitosandsRNA complex and QCH4dsRNA complex were placed on a freshly cleaved mica surface, dried with a
stream of nitrogen, and further dried in an electronic dry cabinet at 25 C for 30 min. All samples were determined by atomic
force microscope (SPA400, Seiko, Japan) machine used the scanner
range 101000 nm area in tapping mode using a Micro cantilever
with 28 kHz resonance frequencies and a constant force, range of
1.41.8 N/m. All images were recorded in air at room temperature
and a scan speed of 0.8 Hz and the phase image and topology used
to determine the morphology and particle size.
2.4. Transfection of dsRNA into Sf9 cells using Cellfectin
(Invitrogen, USA) and chitosan nanopolyplexes
Sf9 cells were cultured in 2 ml of Sf-900 III SFM containing 1% antibiotic-antimycotic (pH 6.2) at a concentration of
5 105 cells/ml for 6-well plate (Costar, Corning, USA) and incubated at 28 C overnight or until 6070% cell conuency. Using
5 l Cellfectin , 2 g of dsRNA were transfected into Sf9 cells
by Cellfectin (Gibco Invitrogen, USA). The treated Sf9 cells were
incubated at 28 C for 12 h prior YHV infection. YHV stock was
prepared from hemolymph of YHV-infected shrimps as previously
described (Saksmerprome et al., 2009). Sf9 cells were infected with
YHV at a multiplicity of infection (MOI) of 20 or around 2 107
copies/well by incubating on a shaker at 28 C for 6 h. In the case of
chitosandsRNA complex, 10 l of complex which contains 2 g of
dsRNA was diluted in 1 ml Sf-900 III SFM without antibiotics prior
to transfection.
Cells were harvested at 12 h after transfection by centrifugation
at 2200 g for 5 min. Total RNAs were extracted by Tri-pure reagent
(Roche, USA). RNA concentrations were determined by UV spectroscopy at OD260 . First strand cDNA was prepared from 150 ng/l
of total RNA using Titan one tube reverse transcriptase (Roche,
USA) at 50 C for 30 min. Double-stranded RNA product in each
RNA sample was monitored by specic primer YHV invert F sal
(5 -ACGCGTCGACGCATGTCCTGTTCTC-3 ) and YHV reverse R pst
Actin-specic
primer
(5 -TTGACGTCGAATTCTAGCCATGC-3 ).
set, Actin-Sf9-Forward (5 -GATATGGAGAAGATCTGGCAC-3 ) and
Actin-Sf9-Reverse
(5 -ACGGGTCTGTTCCCTATGAAGCACCAC-3 ),
was added in PCR reaction for internal control. PCR conditions were
as followed: the initial denaturing step at 94 C, 2 min; denaturing
94 C, 30 s, annealing temperature depending on primers, 30 s,
extension at 72 C, 30 s for 30 cycles; and a nal extension at
72 C, 5 min. PCR products were analyzed using 1.5% agarose gel
electrophoresis.
2.5. Preparation of the FITC-conjugated QCH4 and transfection

2.3. Formation of chitosandsRNA and QCH4dsRNA

nanopolyplexes
Using the recombinant plasmid with YHV RdRp hairpin gene,
dsRNA was prepared in E. coli HT115 (DE3) as previously described
(Saksmerprome et al., 2009). One microgram of chitosan and

Fifty milligrams of QCH4 (0.3 mmol) were dissolved in deionized

water (5 ml). Twelve milligram of uorescein isothiocyanate (FITC,
0.1 equiv./GlcN) was dissolved dimethylsulfoxide (DMSO, 3 ml) and
adjusted to pH of 3.0 with 1 M HCl. Six milligram of 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC, 0.1 equiv./GlcN) was

G. Theerawanitchpan et al. / Journal of Biotechnology 160 (2012) 97104

99

Fig. 1. ChitosandsRNA and QCH4dsRNA complexes at various weight ratios. At weight ratios of chitosandsRNA 1.5 and QCH4dsRNA 0.2 were selected to study knockdown
efciency.

then added and stirred at room temperature for 1 h. Then 3.5 mg of

N-hydroxysuccinimide (NHS, 0.1 equiv./GlcN) was added. The reaction mixture was stirred at room temperature for another 15 min.
Subsequently, the QCH4 solution was added into the reaction mixture and stirred at room temperature for 6 h. Finally, the mixture
was dialyzed against distilled water for 2 weeks and lyophilized.
The obtained FITC conjugated QCH4 was dissolved in water and
used to form nanopolyplexes with dsRNA at the ratio of 10 l
QCH4-FITC: 2 g dsRNA. Transfection of FITC-labeled QCH4 and
FITC-labeled QCH4dsRNA nanopolyplex were carried out, in the
same manner as those of unconjugated chitosan complexes, and
collected at 6, 12, and 24 h after transfection.

trypan blue at dilution 1:10. A drop of the trypan blue/cell mixture

was applied to a hemacytometer. Unstained (viable) and stained
(nonviable) cells were counted separately in the hemacytometer.
Viable cells were calculated as follows.
3. Results
3.1. Synthesis and characterization of QCH4

Fi = uorescence intensity of individual cell was measured by Olympus Fluoview version 1.7 viewer program (FV1000, Olympus,
Japan); i = 1, 2, 3, . . ., n; n = number of analyzed cells.

Quaternization of chitosan was performed by reacting chitosan

with commercially available Quat 188, and iodine was used as
a catalyst in a heterogeneous process under alkaline condition,
yielding water-soluble QCH4. Under this condition, Quat 188 readily generated the corresponding epoxide, which reacted with the
primary amino groups and hydroxyl groups of the chitosan in
a nucleophilic substitution pathway to introduce the quaternary
ammonium moieties (Supplementary Fig. 1 for schematic diagram).
Using 1 H NMR technique described in Sajomsang et al. (2009a,b),
the degree of quaternization (DQ) was determined to be 57%. The
ATR-FTIR spectrum of QCH4 was similar to those of chitosan except
for the presence of an absorption band at 1480 cm1 due to C H
symmetric bending of the methyl groups on the quaternary ammonium substituents (Supplementary Fig. 2). The 1 H NMR spectrum
of QCH4 exhibited characteristic resonances at 4.2, 3.3, 3.2, and
2.6 ppm which were attributed to methine proton, methylene protons, N,N,N-trimethyl protons, and methylene protons, respectively
(not shown). The molecular weight average of the native chitosan
was found to be Mn 48 kDa, Mw 276 kDa and Mw /Mn 5.67 while the
molecular weight average of the QCH4 was Mn 63 kDa, Mw 516 kDa
and Mw /Mn 8.19, respectively. A relatively wide molecular weight
distribution with a polydispersity index (PDI) of chitosan and QCH4
were observed. The quaternization of chitosan led to a increase
in the molecular weight. This was due to quaternary ammonium
moiety introduced to chitosan backbone.

2.7. Cytotoxicity test

3.2. Physicochemical analysis and complex formation

Toxicity test of chitosandsRNA complexes was evaluated by

trypan blue exclusion assay using hemacytometer. Treated cells
were collected at approximately 60 h after seeding and mixed with

Gel retardation analysis suggested that 368-bp dsRNA were

incorporated in the complexes at several weight ratios via electrostatic interaction (Fig. 1). Physicochemical properties of complexes

2.6. Evaluation of dsRNA transfection by immunohistochemistry

analysis
At 12 and 24 h after infection, cells were collected for immunohistochemistry assays. The experiments were carried out using
monoclonal antibodies (MAb) specic for the YHV structural protein gp64 (MAbY18) as previously described (Soowannayan et al.,
2003). Fluorescence intensity representing level of YHV expression
was quantied by Olympus Fluoview version 1.7 viewer programs
(FV1000, Olympus, Japan). Approximately 50150 cells in each
group were analyzed. Statistic difference was determined using
one-way analysis of variance (ANOVA), followed by an LSD post
hoc test with signicance set at p-values of <0.01. Average of YHVantigen uorescence intensity of each treated group (Favg ) was
calculated as follows.
Favg =

i=n Fi
F1 = F2 + F3 + + Fn
=
n
n

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G. Theerawanitchpan et al. / Journal of Biotechnology 160 (2012) 97104

Fig. 2. Physicochemical property and morphology of complexes. (A) Size (), PDI () and zeta potential () of chitosan (QCH4)dsRNA complexes at different weight ratios.
(B) AFM images of (a) naked dsRNA, (b and c) Chitosan and its derivative, QCH4, (d) chitosandsRNA complex at weight ratio of 1.5 and (e) QCH4dsRNA complex at weight
ratio of 0.2.

including size, zeta potential and polydispersity index (PDI) were

measured by using Zetasizer Nano ZS. In Fig. 2A, the size of
chitosandsRNA complexes at weight ratios 1.3, 1.5 and 1.7 were
in the range of 350650 nm, while QCH4dsRNA complexes at
weight ratios 0.16, 0.20 and 0.24 were approximately 150350 nm.

The zeta potential depends on the positive or negative charge

of the complexes. Electronegative values of zeta potential were
observed at weight ratios of 1.3 of chitosandsRNA complex and
0.16 and 0.2 of QCH4dsRNA complexes. At weight ratio of 1.5 of
chitosandsRNA and 0.24 of QCH4dsRNA, the zeta potential was

Fig. 3. RT-PCR analysis. YHV-specic dsRNA were transfected in Sf9 cells using chitosan and its derivative (QCH4) as dsRNA carriers. Symbols a, b and c represent different
weight ratios of a carrier:dsRNA. In the case of native chitosan complex, a = 1.3, b = 1.5, and c = 1.7. For QCH4:dsRNA, shrimp actin was used as internal control for normalizing
RNA level. M: 2 log DNA marker, m: RNA from Sf9 cells without transfection, CF: RNA from dsRNA-transfected cells using Cellfectin as a transfectant and -: negative control
using DEPC water instead of RNA template.

G. Theerawanitchpan et al. / Journal of Biotechnology 160 (2012) 97104

approximately neutral while electropositive value was found in

chitosandsRNA at weight ratio of 1.7.
Polydispersity index of chitosandsRNA complexes at weight
ratios of 1.3, 1.5 and 1.7 were 0.56 0.04, 0.64 0.05 and
0.61 0.09 and QCH4dsRNA complexes at weight ratios of 0.16,
0.2 and 0.24 were 0.59 0.03, 0.52 0.06 and 0.56 0.08 respectively. This result indicated the narrow PDI which implied the
narrow size distribution of nanopolyplexes were obtained. The
morphology of each was monitored under AFM (Fig. 2B). The dsRNA
was condensated as rod-like structure at state equilibrium. The
structures observed with AFM revealed that both chitosan and
QCH4 depict with a brush-like conformation where aggregates
comprise a dense. AFM images of the chitosan complex at 1.5:1
(w/w) and QCH4dsRNA complex at 0.2:1 (w/w) were shown in
Fig. 2d and e, respectively. In a narrow scan of the nanopolyplexes,

101

many spherical-shaped structures of similar size were observed.

The diameter of QCH4dsRNA structure was within 100300 nm.
The size of the chitosan/dsRNA structure was ranging between 300
and 700 nm. The results suggested that the QCHdsRNA complex
was more tightly condensed than the native chitosandsRNA.
3.3. Evaluation of Cellfectin and chitosan nanoparticles as
dsRNA-delivered reagents
Cytotoxicity of Cellfectin , chitosan and QCH4 in Sf9 cells was
investigated using trypan blue exclusion method. As shown in
Table 1, untreated control, chitosan- and QCH4-treated group
exhibited comparable percentage viability (7580%), and the group
transfected with the commercial liposome complex exhibited the
lowest percent viability (65%).

Fig. 4. (A) Average YHV-antigen uorescence intensity from immunohistochemical staining of Sf9 cells using antibodies to structural protein of gp64 YHV. Cells were collected
at 12 (black bar) and 24 hpi (white bar) for determining the level of YHV infection. Error bars indicate the standard deviation. * and ** indicate signicant differences from
the pair-wise comparisons relative to the YHV-infected group at 12 and 24 hpi (p < 0.01), respectively. A.U. arbitrary unit. Immunohistochemistry assay of YHV-infected
cells at 12 (B) and 24 (C) hpi using a monoclonal antibody against YHV structural protein. Positive reactions are shown in red. (a) YHV infection only; (b) Cellfectin -dsRNA
transfection; (c) chitosandsRNA; (d) QCH4dsRNA. (For interpretation of the references to color in this gure legend, the reader is referred to the web version of this article.)

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Fig. 4. (Continued ).

Capability of Cellfectin , chitosan and QCH4 to deliver dsRNA

into Sf9 cells was examined. Total RNAs were extracted from
treated cells after 12 h incubation with complexes. RT-PCR products of 400 bp, indicating the presence of YHV-specic dsRNA,
were detected in the cells incubated with all types of complexes
(Fig. 3). For quantitative data, average uorescence intensity representing quantity of YHV was calculated by FV10-ASW 1.7 Viewer
program. Inhibitory effects by chitosandsRNA and QCH4dsRNA
complexes were not as potent as to the lipid-based tranfectant,
Cellfectin at 12 h after viral challenge (Fig. 4AB). However, signicant (at least 50%) reduction in YHV expression in the groups
treated by the chitosan complexes became evident at 24 h post
infection (Fig. 4AC). We used confocal microscopy for directly
visualizing cellular uptake of the FITC-labeled complex after

Table 1
Percentages of cell viability under different treatments determined by the trypan blue exclusion method. There are no signicant differences in all four groups
(p < 0.01).
Treatments

% cell viability (SD)

Untreated
Cellfectin -dsRNA
ChitosandsRNA
QHC4dsRNA

78.4 (3.2)
64.9(7.9)
74.0 (3.2)
73.9 (4.1)

transfection in Sf9 cells. FITC-labeled QCH4 was selected to form

complex with dsRNA via electrostatic interaction. As shown in
Fig. 5, the green signals for the FITC-labeled QCH4dsRNA complex
were visible throughout 624 h after transfection, and the labeled
complex clearly accumulated at 24 h after transfection. Both RTPCR and confocal immunouorescence microscopy using anti-YHV
antibody revealed utility of chitosan and QCH4 for successful dsRNA
delivery in Sf9 cells for antiviral application.

4. Discussion
In the present work, dsRNA-mediated inhibition of shrimp viral
infection was investigated in Sf9 cells. Previous study by Sriton et al.
(2009) demonstrated that shrimp viruses, including YHV, can be
persistently maintained in Sf9 insect cell line. YHV can infect Sf9
cells, and its antigen is detected by immunohistochemistry using
monoclonal antibody (Y18) against antigen of envelope protein
(gp64) of YHV. General morphology of infected cells is not different from that of uninfected cells or mock cells under phase contrast
microscopy. Taken advantages of reproducibility and convenience,
shrimp virus-infected Sf9 cells can be used to screen for effective
dsRNA carriers, providing new approaches for RNAi applications in
shrimp.

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103

Fig. 5. Confocal images of Sf9 cells incubated with FITC-labeled QCH4dsRNA complex at varying times. Upper and lower panels represent DIC (differential interference
contrast) images, and uorescent-FITC images, respectively. Red arrow indicates accumulation of FITC-labeled complexes in the cell. (For interpretation of the references to
color in this gure legend, the reader is referred to the web version of this article.)

Natural chitosan is shown to form complexes with siRNA and

plasmid DNA for gene silencing (Howard et al., 2006; Liu et al.,
2007) and gene therapy-related studies (MacLaughlin et al., 1998;
Weecharangsan et al., 2008). In this study, chitosan derivative
(QCH4) was prepared from quaternization of native chitosan to
improve water solubility of chitosan by adding positive charges on
chitosan backbone. Both chitosan and QCH4 are shown to mediate
efcient dsRNA delivery into insect cells. QCH4 is more water soluble in a wider range of pH, and it can be used in a lesser amount
relative to natural chitosan for efcient delivery of dsRNA. Doublestranded RNA delivered by QCH4 was detected by RT-PCR within
24 h after transfection (Fig. 3). Confocal images of FITC-labeled
QCH4dsRNA complex indicated the presence of the complex in
cells within 24 h post transfection (Fig. 5). The results from both
detection methods demonstrate that chitosan nanoparticle, such
as QCH4, can be used for effective delivery of long dsRNA in Sf9
cells.
Silencing efciency of chitosandsRNA, QCH4dsRNA complex
and Cellfectin -dsRNA was monitored by immunohistochemistry
assay. In all cases, YHV-specic dsRNA targeting RdRp gene was
delivered and was able to reduce YHV infection (Fig. 4). The result
was in agreement with previous studies in shrimp (Saksmerprome
et al., 2009; Tirasophon et al., 2005) that dsRNA targeting nonstructural genes effectively inhibited YHV replication. Inhibitory
effects by dsRNA in cells treated with chitosan and QCH4 complexes
were not potent at 12 h post infection (hpi) when compared to the
group transfected with Cellfectin complex. In contrast, at 24 hpi
silencing efciency of the chitosan complexes was more prominent
than that of the commercial lipoplex. Deviation of effective time
of Cellfectin and chitosan is probably resulted from their release
properties. Cellfectin is a cationic-lipid formulated for binding
nucleic acid via adsorption on the lipid surface, while chitosan
complexes occurred through electrostatic interaction. After internalization of complex into the cell, the release of dsRNA from the
chitosan complex is probably more difcult because dsRNA is well
condensed with polymer to form particles.
In conclusion, this study demonstrates the effective use of insect
cells infected with shrimp virus for selection of dsRNA carriers

in RNAi-mediated application. Long dsRNA (>100 bp) targeting

YHV, formulated with chitosan and its quaternized derivative,
has been shown to inhibit YHV propagation in Sf9 cells with
minimal cytotoxicity. Our results show that cellular uptake of chitosan nanopolyplexes was correlated to RNAi-mediated efciency.
Chitosan-based nanoparticles can be used for dsRNA delivery
in insect culture as inexpensive transfection reagents with low
cytotoxicity. Ultimately, these nanopolyplexes will be further
investigated if they could serve as orally delivered agents for development of antiviral feed.
Acknowledgments
The authors would like to thank Dr. Paisan Sithikorngul
(Srinakharinwirot University) for providing primary antibody targeting YHV, and Dr. Suparerk Borwornpinyo (Mahidol University)
for cell culture training to GT. The work was supported by grants
from Thailand Graduate Institute of Science and Technology (TGIST)
and International Foundation for Science (IFS) to GT and VS, respectively.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.jbiotec.
2012.04.011.
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