J Nanopart Res (2008) 10:151–162

DOI 10.1007/s11051-007-9233-7

RESEARCH PAPER

Hydrophobically modified chitosan/gold nanoparticles for

DNA delivery
Shanta Raj Bhattarai Æ Remant Bahadur K.C. Æ
Santosh Aryal Æ Narayan Bhattarai Æ
Sun Young Kim Æ Ho Keun Yi Æ Pyoung Han Hwang Æ
Hak Yong Kim

Received: 31 January 2006 / Accepted: 2 April 2007 / Published online: 4 May 2007

Springer Science+Business Media B.V. 2007

Abstract Present study dealt an application of mod-
ified chitosan gold nanoparticles (Nac-6-Au) for the
immobilization of necked plasmid DNA. Gold nano-
particles stabilized with N-acylated chitosan were
prepared by graft-onto approach. The stabilized gold
nanoparticles were characterized by different physico-
chemical techniques such as UV-vis, TEM, ELS and
DLS. MTT assay was used for in vitro cytotoxicity of
the nanoparticles into three different cell lines (NIH
3T3, CT-26 and MCF-7). The formulation of plasmid
DNA with the nanoparticles corresponds to the complex
forming capacity and in-vitro/in-vivo transfection
S. R. Bhattarai
Remant Bahadur K.C.
S. Aryal
Department of Bionanosystem Engineering, Chonbuk
National University, Chonju 561-756, Republic of Korea
N. Bhattarai
Department of Materials Science and Engineering,
University of Washington, Seattle, WA 98195, USA
S. Y. Kim
P. H. Hwang
Department of Pediatrics, School of Medicine, Chonbuk
National University, Chonju 561-756, Republic of Korea
H. K. Yi
Department of Biochemistry, School of Dentisty,
Chonbuk National University, Chonju 561-756, Republic
of Korea
H. Y. Kim (&)
Department of Textile Engineering, Chonbuk National
University, Chonju 561-756, Republic of Korea
e-mail: khy@moak.chonbuk.ac.kr
efficiency was studied via gel electrophoresis and
transfection methods, respectively. Results showed the
modified chitosan gold nanoparticles were well-dis-
persed and spherical in shape with average size around
10*12 nm in triple distilled water at pH 7.4, and
showed relatively no cytotoxicity at low concentration.
Addition of plasmid DNA on the aqueous solution of
the nanoparticles markedly reduced surface potential
(50.0*66.6%) as well as resulted in a 13.33% increase
in hydrodynamic diameters of the formulated nanopar-
ticles. Transfection efficiency of Nac-6-Au/DNA was
dependent on cell type, and higher b-galactosidase
activity was observed on MCF-7 breast cancer cell.
Typically, this activity was 5 times higher in 4.5 mg/ml
nanoparticles concentration than that achieved by the
nanoparticles of other concentrations (and/or control).
However, this activity was lower in in-vitro and
dramatically higher in in-vivo than that of commercially
available transfection kit (Lipofectin1) and DNA.
From these results, it can be expected to develop
alternative new vectors for gene delivery.
Keywords Chitosan
DNA delivery
Gene therapy

Gold nanoparticles
Non viral vectors
Nanomedicine
Introduction
Gene therapy holds an excellent means for curing
acquired and inherited diseases in a straightforward

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way by adding, correcting, and replacing the affected
genes. Two major delivery systems have been used in
the current gene therapeutic approaches viz: viral and
non-viral mediated system (Lundstrom 2003; Ana
et al. 2002). Viral-mediated systems are the most
effective means for delivery and expression of gene.
However, such use is not so frequent due to some
sever limitations like: restricted immunogenicity,
pathogenicity, targeting efficiency etc in their
in vivo and in vitro use. The need of current
methodology is to attribute these limitations (Tripa-
thy et al. 1996). Hence, despite their comparatively
low efficiency, non-viral mediated systems have
attracted a great deal of interest in this field. Efficient
delivery of therapeutic genes into the target cells;
in vitro and in vivo is the major limitation of non-
viral mediated gene therapeutic approaches (Tripathy
et al. 1996). Non-viral mediated gene transfer vehi-
cles with appropriate functional groups, which are
protonated at physiological pH, have been employed
as an effective carrier due to their excellent electro-
static interaction with therapeutic genes (Koping-
Hoggard et al. 2001; Ferrari et al. 2002; Ruponena
et al. 2003). Many attempts have been performed for
the betterment of gene delivery using non-viral
vectors viz: biomolecules, natural polymers, synthetic
polymers etc (Schuber et al. 1998; Mao et al. 2001;
Ravi Kumar et al. 2004).
In recent years, potentiality of chitosan as a non-
viral gene carrier has been extensively considered (Roy
et al. 1999). In acidic pH, the protonated amino groups
of chitosan and chitosan-based materials can effec-
tively bind to DNA and condense it as nano/micropar-
ticles (Lee et al. 1998; Leong et al. 1998; Maclaughlin
et al. 1998; Ishii et al. 2001). Chitosan microparticles
containing reporter genes are being extensively used
for the transfection of mammalian cells both in vitro
and in vivo conditions (Corsi et al. 2003; Iqbal et al.
2003). However, the use of chitosan and chitosan-
based materials as a gene carrier remains inadequate
due to uncontrolled size and inappropriate processing
media (insoluble in physiological pH). So, modifica-
tion (chemical and physical) of natural chitosan is
supposed to be an excellent means for the formulation
of better gene delivery vehicle. Various approaches
viz: modification with ligands (Mao et al. 2001; Kim
et al. 2004; Park et al. 2001; Thanou et al. 2002),
blending with polymers; poly-l-lysine (Aral and
Akbuga 1999; Quong et al. 1999; Quong and Neufeld
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1998) have been frequently performed for the formu-
lation of effective chitosan and chitosan-based mate-
rials to enhance the efficiency of gene delivery.
Recently, hydrophobically modified chitosan has also
been used in gene delivery (Chae et al. 2005; Kai et al.
2004). On the other hand, many clinical studies with
pure elemental gold are just getting underway which
employ microscopic particles of this inert metal as a
vehicle for gene delivery (Kulmeet et al. 2002). Pre-
clinical studies have established that naked DNA
(including defined gene sequences) can be adsorbed
to the surface of minute metallic gold particles and
efficiently delivered by a controlled helium pulse to the
cells of inferior epidermis (Pertmer et al. 1995). It has
been undertaken to evaluate the potential technological
risks attributed to gold itself and to anticipate any
possible complexities which may arise from the
application of this promising new approach to gene
therapy. However, the use of gold as gene carrier in an
aqueous medium has several limitations because of its
rapid aggregation.
Generally, most gene delivery strategies have
focused on the parenteral route of delivery, and oral
administration has been largely ignored due to the
large hurdles that need to be overcome for gene
delivery, such as acidity in stomach, the nuclease,
lipases and peptidases present in the gastrointestinal
tract, and poor permeability of both genes and gene
vectors across the intestinal epithelium owing to the
size and charge of gene delivery vehicles. Inorganic
nanoparticles (silica or gold) is an inert materials with
no obvious sensitivity with acid pH and intestinal
digestive enzymes, and chitosan is a natural biode-
gradable and biocompatible mucoadhesive polysac-
charide that has been widely used in oral gene
delivery (Roy et al. 1999). Moreover, Chitosan also
increases the transcellular and paracellular transport
across mucosal epithelium (Artursson et al. 1994),
further indicative of its potential in oral gene delivery
and in generating protective mucosal immune
responses.
Realizing their potential application in gene
delivery, we already explored formulation procedure
of chitosan and gold so as to overcome their
limitation. The beauty of our formulation was
significant stability of N-acylated chitosan stabilized
gold nanoparticles in physiological condition. Here,
the N-acylated chitosan play dual roles as a stabilizer
and a carrier. On the other hand, gold particles
J Nanopart Res (2008) 10:151–162
provide the nanoscopic, monodisperse nanoparticles,
and act as contrast agent while detecting delivery site.
However, current study describes hydrophobically
modified chitosan stabilized gold nanoparticles as
a novel DNA carrier for gene delivery, in-vitro and
in-vivo.
Experimentals
Instrumental
UV-vis absorption spectra of the samples were
recorded in Cary 500 UV-vis-NIR spectrometer.
Particle size and morphology were observed by
JEOL JEM 2010 transmission electron microscope
(TEM) operating at 200 kV. Samples for TEM were
prepared by dipping a carbon-coated copper grid in
an aqueous dispersion of nanoparticles and dried at
room temperature. Particle size and its distribution
was determined using dynamic light scattering (DLS)
(Malvern System 4700) equipped with vertically
polarized light supplied with argon-ion laser (Cyon-
ics) with measuring angle of 908 to the incident beam.
f-potential of the nanoparticles was determined by
electrophoratic light scattering (ELS) (ELS 8000/
6000 Otsuka electronics Co., Japan) with measuring
angle of 208 to incident beam. Each measurement
Fig. 1 Chemical structure
chitosan. Subscripts m and
n represent the variable
number 78 and 22
respectively
153
was performed at room temperature after sonicating
the samples into an ultra-sonicator bath for 1 min.
Reagents
Chitosan-10 (viscosity average molecular weight,
Mv = 2.1 · 105, degree of deacetylation 78%) was
purchased from Wako Pure Chemical Industries,
Ltd., Japan. Viscosity average molecular weight of
chitosan was determined according to the previous
report (KC et al. 2006). Fatty acyl chlorides (e.g.,
hexanoyl chloride and octanoyl chloride), hydrogen
tetrachloroaurate (HAuCl4), sodium borohydride
were purchased from Sigma-Aldrich Co., and used
without any further purification. All other chemicals
were purchased from Showa chemical Ltd. of Korea.
Preparation of self-assembled N-acylated
chitosan/gold (Nac-6-Au) nanoparticles
Chemical structure of native and N-acylated chitosan
is shown in Fig. 1. Hydrophobic modification of
native chitosan i.e. the preparation of N-acylated
chitosan (Nac) was done using different fatty acyl
chlorides (Le et al. 2003). Grafting of Nac on gold
nanoparticles (Nac-Au) was taken from previous
publication (KC et al. 2006). Briefly, freshly prepared
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HAuCl4 aqueous solution (10 mM, 1.0 ml) was added
to the 2.0 ml polymer solution (33% in 0.1 M HCl)
and stirred for 1 h. To this solution, freshly prepared
ice-cold sodium borohydride (0.1 M, 0.4 ml) was
added under moderate stirring at room temperature.
Rapid color change to pink indicates the formation of
gold nanoparticles. Thus formed gold nanoparticles
were purified and collected using ultracentrifuge
operated at 35,000 g for 30 min at 48C. Here two
types of Nac-Au (Nac-6-Au, Nac-8-Au) have been
formulated, out of which Nac-6-Au was selected for
the DNA delivery due to its higher stability (KC et al.
2006).
Plasmid amplification
The procedure for plasmid amplification was taken
from our previously published report (Bhattarai et al.
2003). Briefly, plasmid DNA (pcDNA3.1His/Myc/
LacZ) (Invitrogen, USA) with a size of 6.6 kb
containing bacterial b-galatosidase gene (LacZ with a
size of 1.2 kb) as the reporter gene under the control
of CMV (cytomegalovirus) promoter was used in this
study. Escherichia coil (E. coli) JM109 Bacterial
strain was used as host cell for amplification of
plasmids. The transformed cells were grown in large
quantities of LB broth supplemented with Ampicillin
(10 mg/ml). The plasmid DNA was purified by
phenol–chloroform and was diluted in sterilized
water. Purity was conformed by 1% Agarose gel
electrophoresis followed by Ethdium bromide (EtBr)
staining, and DNA concentration was measured by
UV absorption at 260 nm.
Cell line preparation
Cells (NIH 3T3; mouse embryo cell, CT-26; colon
cancer cell and MCF-7; breast cancer cell) were used
for transient transfection experiments and cytotoxic-
ity, and grown at 378C under 5% CO2 atmosphere as
described in our previous report (Bhattarai et al.
2006). The following media were used: 1. Dulbecco’s
modified Eagle’s medium (DMEM) (Gibco) with
10% (v/v) fetal calf serum (Gibco) for CT-26 and
MCF-7 cells, and 2. RPMI-1640 medium containing
with 10% (v/v) fetal bovine serum (FBS) (Gibco) for
3T3 cells. For all media, penicillin (100 U/ml) and
streptomycin (100 lg/ml) was used. During transfec-
tion experiment cells were supplemented with Nac-6-
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Au/DNA complexes, and the plates were slowly
agitated for 2 min, and incubated for 4 h at 378C, 5%
CO2 atmosphere. After 4 h, media was replaced by
fresh media containing 10% FBS, and again incu-
bated in same condition up to 48 h.
Evaluation of cytotoxicity
Evaluation of the cytotoxicity was performed by the
MTT assay in four kinds of cell lines (MCF-7, 3T3
and CT-26 cells). Briefly, various cell suspensions
containing 1 · 104 cell/well in RPMI-1640 for NIH-
3T3 cell and DMEM for MCF-7 and CT-26,
containing 10% FBS were distributed in a 96-well
plates, and incubated in a humidified atmosphere
containing 5% CO2 at 378C for 24 h (Bhattarai et al.
2003). The cytotoxicity of Nac/Au nanoparticles was
evaluated in comparison with control cells. Cells
were incubated for additional 24 h after the addition
of defined concentration of Nac/Au nanoparticles.
The mixture was replaced with fresh medium
containing 10% FBS. Then, 20 ll of MTT solution
(5 mg/ml in 1 · PBS) were added to each well. The
plate was incubated for an additional 4 h at 378C.
Next, MTT-containing medium was aspirated off and
150 ll of DMSO were added to dissolve the crystals
formed by living cells. Absorbance was measured at
490 nm, using a microplate reader (ELX 800; BIO-
TEK Instruments, Inc.). The cell viability (%) was
calculated according to the following equation:
Cell viability (%) = [OD490(sample)/OD490(con-
trol)] · 100.
Preparation of DNA complexes
Nac-6-Au nanoparticles and pcDNA3.1His/Myc/
LacZ plasmid was used for preparation of complexes
in phosphate buffer (PBS, pH 7.4). The plasmid DNA
(5 lg) was mixed with different volume (40–200 ll)
of Nac-6-Au nanoparticles solution from the stock
solution (50 mg/ml) of that nanoparticles with final
volume 1 ml PBS so as the final concentration of the
resulting nanoparticles became 2*10 mg/ml. The
resulting mixture was stored for 30 min at room
temperature and then used in DNA uptake or
transfection experiment. Results were observed by
X-gal staining method, and quantified by b-galacto-
sidase assay.
J Nanopart Res (2008) 10:151–162
Analysis of DNA complexes
DNA complexes corresponds to the DNA binding
with the nanoparticles was first analyzed by spectro-
photometer, and furthermore verified with gel elec-
trophoresis. Samples were prepared as described in
preparation of DNA complexes. Resulting samples
were stored in room temperature for 6 h and then
centrifuge at 13,000 g (revolution per minute) at 48C
for 20 min. About 10 ml of the supernatant from each
samples was taken out and re-diluted in 1 ml
autoclaved triple distilled water for spectrophotome-
ter analysis at OD = 260. Remaining supernatant
portion was discarded, and sedimented portion of
each sample was again diluted with 20 ll autoclaved
triple distilled water and vortexed for 10 min before
loading onto 1% agarose gel for gel electrophoresis
(for band analysis).
Transfection of cells and b-galactosidase assay
Cells were seeded in 24-well plates (5 · 104 cells/
well) and grown at standard culture condition for
24 h. Culture media were changed with fresh
complete media containing defined concentration of
Nac-6-Au/DNA nanoparticles as described in prepa-
ration of DNA Complexes. After 48 h of incubation,
cells were harvested for b-galactosidase assay. The
assay was done as previously described method
(Bhattarai et al. 2006). Briefly, culture media were
discarded and the cells were washed with PBS. The
cells were detached with trypsin, suspended in PBS,
and collected by centrifugation. The cells were lysed
in 200 ll of lysis buffer containing 100 mM KH2PO4/
K2HPO4 (pH 7.4), 0.2% Triton X-100, and 1 mM
DTT by freezing and thawing. The b-galactosidase
assay was performed in a microtiter dish. About 25 ll
of cell lysate was added to 135 ll of buffer containing
100 mM KH2PO4/K2HPO4 (pH 7.4), 10 mM KCl,
1 mM MgSO4, and 50 mM 2-mercaptoethanol, and
incubated for 5 min at 378C. Then, 50 ll ONPG
(O-nitrophenyl-b-d-galactopyranoside) substrate
solution (4 mg/ml ONPG in 100 mM phosphate
buffer, pH 7.4) was added to the reaction mixture and
incubated for 1*16 h at 378C. After the incubation
period, the reaction was terminated by addition of
90 ll stop solution (1 M Na2CO3) and the absorbance
of samples was measured with a microtiter dish
reader set at 420 nm. Protein concentration of cell
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lysate was determined with Bradford method. The b-
galactosidase activity was calculated by using the
following equations, and units of enzyme were
expressed as nanomoles of b-galactose formed per
min. b-galactosidase activity (U/mg of total protein in
lysate) = [OD 420/0.0045 · assay volume (ml)]
min 1 mg 1.
X-gal staining of transfected cell
For X-gal staining corresponds to the expression of
LacZ gene was established after adding the fixing
solution [2% (v/v) formaldehyde, 0.2% (v/v) glutar-
aldehyde and 1 · phosphate buffer (1 · PBS)] on the
transfected cells seeded in 24-well plates
(5 · 104 cells/well) grown at standard culture
conditions for 24 h. After fixing 1 h, the plate was
washed 3 times by 1 · PBS solution and X-gal
staining was performed with X-gal staining solution
(2 mM X-gal, 2 mM K4Fe (CN)6, 2 mM K3Fe (CN)6,
2 mM Mgcl2, 10 · PBS) for overnight at 378C.
Transfection of cells corresponding to the expression
of blue color was monitored by light microscope and
images were digitally photographed. For the compar-
ison purpose commercially available transfection kit,
Lipofectin1 (Invitrogen, USA) was also used during
the transfection study.
In vivo gene expression
Female C57BL/6 mice were purchased from the
Korean Research Institute of Chemical Technology
(Daejeon, Chuungnam, Korea) and were housed in an
environment-controlled rearing system. The mice
were maintained in animal facilities at the Chonbuk
National University and used in accordance with the
guidelines of the University. All mice were used in
experiment at 7–8 weeks of age. The C57BL/6 mice
were fed either Nac-6-Au nanoparticles containing
the LacZ gene (pcDNA-LacZ, 50 mg per mice) or
plasmid DNA (pcDNA-LacZ) with Lipofectin, using
animal feeding needles. Three days later, the mice
were killed and their stomachs and small intestines
were surgically removed. A galacto-Star Kit (Tropix,
Bedford, MA, USA) was used to measure in vivo
reporter gene expression. Briefly, at defined times
after oral delivery, mice were sacrificed, with their
stomachs and small intestines harvested and homog-
enized for 20 s with 1 ml lysis buffer containing
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protease inhibitors cocktail (Boehringer Mannhein,
Germany) and centrifuged at 12,500 g for 10 min at
48C. The supernatant fluid was heated at 488C for
60 min to inactivate endogenous b-galactosidase
activity. The sample was centrifuged again and
measured total protein concentration. Two hundred
micrograms of protein from each sample was mixed
with 70 ll reaction buffer in Monolight Luminometer
cuvettes (Pharmingen, San Diego, CA, USA) and
incubated at room temperature for 60 min. The b-
galactosidase activity is expressed as relative light
units per milligram protein (U/mg).
Results and discussion
Characterization of self-assembled N-acylated
chitosan/gold (Nac-6-Au) nanoparticles
UV-vis spectra of gold hydrosol and Nac-6-Au
nanoparticles showed a characteristic surface plas-
mon band (SPB) at 512, and 541 nm, respectively,
suggesting the formation of gold nanoparticles
(Fig. 2). A significant red shift in the SPB of Nac-6
capped gold nanoparticles (curves B) compared to
gold hydrosol (curve A) suggests a linear increase in
particle size consequent to the surface modification of
particle (Daniel and Astruc 2004; Chakrabarti and
Klibanov 2003; Aryal et al. 2006). Furthermore
characterization of the particles was taken from the
previous publication (KC et al. 2006).
Fig. 2 UV absorbance spectra of; gold hydrosol (A), N-acylated chitosan-gold (Nac-6-Au) (B)
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Physiochemical characterization of Nac-6-Au
nanoparticles with or without DNA
Figure 3 shows the DLS data and TEM photographs
of Nac-6-Au nanoparticles and the nanoparticles with
plasmid DNA. The result of DLS measurement
showed a uni-model size distribution of nanoparticles
without DNA and with DNA. The average size of
Nac-6-Au nanoparticles without DNA was 13.5 nm
where as with DNA was 15.34 nm (Fig. 3a, A, B).
TEM micrograph of Nac-6-Au nanoparticles showed
a well dispersed, spherical and regular nanoparticle
with average size 12.9 ± 0.2 nm (Fig. 3b A). The
shape and regularity of nanoparticles with DNA at
low concentration was not so different from Nac-6-
Au nanoparticles (Fig. 3b, B), but with the increase in
the concentration of DNA the shape of individual
nanoparticles was clustered (Fig. 3b, C) moreover
aggregated, which is one of the hindering factor in
gene delivery. However, the nanoparticles with
plasmid DNA (at low concentration) observed in this
study were relatively small, highly disperse and
suitable for mammalian cells uptake (Fig. 3b, B).
Table 1 shows the f-potential of Nac-6, Nac-6-Au
and Nac-6-Au/DNA. The nanoparticles of Nac-6
being a polycation gives different +ve f–potential
depending on the pH of media. On partial acylation,
f-potential of the Nac-6 was + 50 mV at pH 10 where
as that potential was + 55 mV at pH 7.4. The
f-potential of the Nac-6 was increased up to + 40 mV
at pH 7.4 after incorporation of gold, and further
J Nanopart Res (2008) 10:151–162 157

Fig. 3 Size and size distribution of nanoparticles; Nac-6-Au Transmission electron micrograph (TEM) of Nac-6-Au (b,
(a, A) and Nac-6-Au/DNA (a, B). Size was measured A), Nac-6-Au/DNA (b, B), and Nac-6-Au with higher
using photon correlation spectroscopy (dynamic light scatter- concentration of plasmid DNA (b, C). Scale bar represents
ing, DLS) and data were plotted as number distribution. 30 nm

decreased to + 20 mV at pH 7.4 as complexes with physiological condition (pH 7.4) is still acceptable for
plasmid DNA. Nac-6-Au nanoparticles with or with- transfection of mammalian cells.
out DNA results that the addition of plasmid DNA
increased the hydrodynamic diameter (13.33%) of the Evaluation of cytotoxicity
nanoparticles. Furthermore, it can be inferred that the
f-potential of Nac-6, Nac-6-Au and Nac-6-Au/DNA Cytotoxicity of gene transfection vectors including
depends upon pH of resulting solution, and markedly viral vectors, cationic liposomes and polymeric
reduces (55.5*66.6%) after addition of the plasmid cations is a major barrier to efficient delivery of
DNA at pH (10 and 7.4) (Table 1). However, the exogenous genes. Whether the presently formulated
f-potential of Nac-6-Au with the plasmid DNA at vector (Nac-6-Au nanoparticles) influenced cell via-
bility was investigated in three different cell lines.
MTT assays were performed to evaluate the cytotox-
icity. Figure 4 shows the representative data of cyto-
Table 1 Variation in f-potential (mV) of nanoparticles at toxicities from three different experiments with
different composition increasing concentration of the Nac-6-Au nanoparti-
Samples f -potential (mV) f -potential (mV) cles. The Nac-6-Au nanoparticles at low concentration
(pH 10) (pH 7.4) (<16 mg/ml) showed relatively no significant toxicity
on the cells. The cell viabilities in the presence of
Nac-6a +50 +55
Nac-6-Au nanoparticles suspension ranged between
Nac-6-Aub +30 +40
c 98% and 110% of the control in all experiments. At a
Nac-6-Au/DNA +10 +20
maximum Nac-6-Au nanoparticles concentration
a
N-acylated chitosan (>32 mg/ml), the mean cell viabilities of the three
b
N-acylated chitosan/gold different cell lines showed about 89–96% viability
c
N-acylated chitosan/gold/DNA compared with that of the control. Interestingly, even at

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high concentrations of Nac-6-Au nanoparticles up to
45 mg/ml, which is 10*15-fold higher than the
concentration required for high efficiency of transfec-
tion, Nac-6-Au showed no obvious negative effect on
cell viability.
From cytotoxicity results, it was shown that the
Nac-6-Au nanoparticles suspension was not toxic to
the cell at low concentration. In contrast, at the higher
concentration, it has been investigated that the
cytotoxicity correlates with membrane damage effect.
Most of the polycations can bind to the negatively
charged plasma membrane and destabilize them.
However, the reduction in membrane toxicity in
present study could be due to well dispersability of
Nac-6-Au nanoparticles in aqueous medium conse-
quently suppress the interaction with cell membrane
But, higher concentration (>16 mg/ml) of the Nac-6-
Au nanoparticles may aggregate, and accumulate
around the cell membrane, and interfere the normal
biological process, which may lead the cytotoxic
effect. Moreover, present Nac-6-Au nanoparticles
that may not prolong the cytotoxicity even in high
concentration (<20 mg/ml) because chitosan and gold
are more biocompatible polymer and metal, respec-
tively.
Analysis of DNA complexes
Complex formation between plasmid DNA and the
Nac-6-Au nanoparticles is correlated with DNA
binding with the nanoparticles. Figure 5 shows bar
diagram and gel electrophoresis to determine com-
plex forming capacity corresponds to the DNA
Fig. 4 Cell viability assay. The cell viability was estimated
after 36 h using MTT colorimetric assay. The assays performed
in triplicate and standard error is shown. Error bars repre-
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binding with Nac-6-Au nanoparticles. Bar diagram
represents the results of spectrophotometer with
increasing concentration of the Nac-6-Au nanoparti-
cles from 1.0 mg/ml to 6.0 mg/ml, the absorbance
was significantly decreased and was minimum at
4.5 mg/ml of the particle concentration. Decreased
absorbance means the decreased plasmid DNA in
supernatant corresponds to the binding or complexes
with the nanoparticles and settles down as sediment.
Furthermore, these results were verified by sedimen-
tation analysis using gel electrophoresis. Results were
analyzed on the basis of observation by comparing
the brightness of DNA bands Fig. 4.
Amount of DNA in gel was significantly changed
after adding different concentration (1.0*6.0 mg/ml)
of the Nac-6-Au particles from the stock 50 mg/ml.
At the lower concentration of the nanoparticles
(1.0*2.0 mg/ml), the bright band of DNA was not
significant. It means that the DNA did not interact
with the Nac-6-Au nanoparticles, Fig. 5 (Lanes, 1 to
2). But this band was significantly increased with
increasing concentration of the particles
(2.0*4.5 mg/ml), Fig. 5 (Lanes 3 to 8) and highly
bright (high concentration of DNA) at the concen-
tration of 4.5 mg/ml Nac-6-Au nanoparticles, Fig. 5
(Lane 8). From these two results (spectrophotometer
and gel electrophoresis), we concluded that the Nac-
6-Au nanoparticles at optimum concentration
(4.5 mg/ml) could have complex forming capacity
with the DNA. In our separate experiment, results
from the gel electrophoresis showed that even higher
concentration ( >4.5 mg/ml) of the Nac-6-Au nano-
particles was not destructive for plasmid DNA. It
sent standard deviation (n = 3). Control means the cells
growing in normal condition without adding the Nac-6-Au
nanoparticles
J Nanopart Res (2008) 10:151–162
Fig. 5 Bar diagram and gel electrophoresis represents the
optimum composition of the Nac-6-Au nanoparticles with
constant amount of DNA for complex formation corresponds to
the binding activity with plasmid DNA. Error bars represent
standard deviation (n = 3). Different concentration of the Nac-
6-Au nanoparticles from 1.0 mg/ml to 6.0 mg/ml was added
into the constant amount of the plasmid DNA (5 lg). The
resulting solution was vortex for 10 min and kept for 6 h at
room temperature before centrifuge (13,000 g/48C) for 15 min.
From each sample, supernatant solution was used for
spectrophotometer analysis and sediment part was used for
means, the present nanoparticles may increase the
bioavilability of plasmid DNA for in vivo applica-
tion. Furthermore, it has been suggested that the
efficacy of transfection with complexes formed
between DNA and cationic polymers strongly
depends upon the complex composition. That’s
why, this paper studies the complexes having the
optional composition of the nanoparticles (4.5 mg/
ml) was shown to be most effective for transfection
on three different cell line (3T3, CT 26 and MCF-7).
However, data shown here is only one cell line
(MCF-7) because of its higher b-galactosidase activ-
ity compared to the other cell lines (3T3 and CT 26).
Optimization of DNA delivery and
b-galactosidase assay
Figure 6 shows the transfection efficiacy using
b-galactosidase assay on MCF-7 cells with different
concentration of the nanoparticles with fixed
concentration of plasmid DNA (5 lg). High internal-
ization (plasmid DNA uptake) corresponds to the
higher value of b-galactosidase activity, which was
159
gel electrophoresis. For gel electrophoresis, all samples were
run on a 1% agarose gel and stained with ethidium bromide
(EtBr). Marker means Hind III and control means pure plasmid
DNA with out Nac-6-Au nanoparticles. Lanes (1*10) contain
the Nac-6-Au nanoparticles concentration 1.0, 1.5, 2.0, 2.5, 3.0,
3.5, 4.0, 4.5, 5.0 and 5.5 mg/ml with constant amount of the
plasmid DNA (5 lg). Lanes (1*2) shows almost lack of
plasmid DNA where as lane (8) shows maximum plasmid
DNA. Over all visual bands also indicate that there was not
destructive interaction between Nac-6-Au nanoparticles and
plasmid DNA
significantly increased when the plasmid DNA mixed
with different concentration of the N-acylated chito-
san gold (Nac-6-Au) nanoparticles (1.0* 6.0 mg/ml)
on MCF-7 cell. At optimum concentration (4.5 mg/
ml) of the nanoparticles, the internalization of
plasmid DNA uptake was about 5 folds higher than
that observed in other concentrations (or/and control)
which was a correct composition of DNA/nanopar-
ticles complexes as shown in Fig. 5 (Lane 8). The
presence and absence of serum in the transfection
medium did not affect the transfection efficiency
(data not shown).
Transfection on MCF-7 cells is our promising
result; so far we are unable to predict the mechanism
of action of the present nanoparticles, which remains
to be further explored. However, there may be some
possibility that present nanoparticles may probably
involve an important role, either interaction with the
cell membrane resulting in the nonspecific changes
on membrane properties (such as ion transport
potential and possibly fluidity) or destabilizing the
endosomal environment. Furthermore, the present
nanoparticles may bind to cells via their net positive
123
160
charge and the adhesion being improved by the
interaction between the positively charged complexes
and the negatively charged cell membranes as well as
minimized the particle aggregation in buffers, spe-
cifically in the transfection medium. Based on this
hypothesis, an increased transfection efficiency of
Fig. 6 can be interpreted.
Furthermore, we compared the transfection effi-
ciency of Nac-6-Au nanoparticles at 4.5 mg/ml
concentration with the commercial transfection
reagents (Lipofectin1, 1 mg/ml) on MCF-7 breast
cancer cells, Fig. 6 (photographs, A and B). Higher
transfection efficiency corresponds to the number of
blue colored cell was clearly seen in both photo-
graphs. But this number was 40*50 times lower in
Nac-6-Au nanoparticles transfected cells than that
observed by the complex of commercially available
transfection reagents (Lipofectin1). Our studies did
not optimize different conditions like time courses for
maximum gene expression, pH of media solution etc.
However, in limited condition, the present Nac-6-Au
nanoparticles showed suitable carrier for gene deliv-
ery, in vitro. For its better use, furthermore conditions
should be optimized.
To assess the expression and distribution of
transduced genes after oral DNA delivery, we fed
Fig. 6 Bar diagram represents the transfection efficacy using
b-galactosidase assay on MCF-7 cells with different concen-
trations (1.0*6.0 mg/ml) of the Nac-6-Au nanoparticles with
constant amount of plasmid DNA (5 lg). b-gal reporter gene
activity is presented as light units per mg of proteins. Error bars
represent standard deviation (n = 3). Photographs (A and B)
123
J Nanopart Res (2008) 10:151–162
C57BL/6 mice either Nac-6-Au/DNA nanoparticles
containing the LacZ gene or plasmid DNA (LacZ)
with Lipofectin1. We determined the tissue expres-
sion of bacterial b-galacotosidase (LacZ) in the
stomach and small intestine 3 days after the oral
administration (Fig. 7). The activity sections repre-
sent, on average, 50% of the whole small intestine.
Although naive mice and mice fed Lipofectin/DNA
showed some activity, mice fed the Nac-6-Au/DNA
nanoparticles showed a higher level of gene expres-
sion in both the stomach and small intestine. We
further compared this activity and found highly
expression in intestine compared to stomach. Inter-
estingly, b-galactosidase activity was 15*20 times
higher expression with Nac-6-Au/DNA nanoparticles
compared to the Lipofectin method. Although the
histological sections of the whole tissue remains to be
illustrated to see the staining patterned as well as
distribution of delivered gene in or around the
epithelial cells (both the stomach and small intestine).
In contrast to in-vitro, presently formulated Nac-6-
Au/DNA system seems to be highly applicable in in-
vivo especially to oral gene delivery. The reason
behind it would be inorganic nanoparticles (gold) is
an inert materials with no obvious sensitivity with
acid pH and intestinal digestive enzymes, and
represent the comparison of transfected cell of MCF-7 between
Nac-6-Au nanoparticles (A) and commercial lipofectamine1
(B). Higher transfection efficiency corresponds to the number
of blue colored cells were observed in photographs of MCF-7
cells with light microscope
J Nanopart Res (2008) 10:151–162
Fig. 7 Bar diagram represents the transfection efficacy using
b-galactosidase activity in stomach and intestine of BALB/C
mice with or without Nac-6-Au nanoparticles. b-gal reporter
chitosan is a natural biodegradable and biocompatible
mucoadhesive polysaccharide. Moreover, Chitosan
also increases the transcellular and paracellular
transport across mucosal epithelium (Artursson
et al. 1994), further indicative of its potential in oral
gene delivery and in generating protective mucosal
immune responses.
Conclusion
A stable and reproducible formulation of Nac-6-Au
nanoparticles has been obtained via surface modifi-
cation of gold nanoparticles. It was adopted by
grafting N-acylated chitosan on the surface of gold
nanoparticles that ensures the physico-chemical sta-
bility in aqueous medium at physiological pH 7.4.
Nac-6-Au/DNA nanoparticles complexes were pre-
pared under defined conditions. The size of the N-
acylated chitosan gold nanoparticles (Nac-6-Au)
(after and before complex formation) was optimized
to be in a nano-size range. f-Potential of these
particles/complexes was varied according to the pH.
Aqueous solution of the Nac-6-Au nanoparticles had
ability to form complexes with plasmid DNA through
electrostatic interaction, and considerable size and f-
potential in physiological (pH 7.4) for DNA delivery.
Above all characteristic feature suggest that chitosan
or chitosna base stabilized gold nanoparticles could
be a suitable vector for oral gene delivery. At present
161
gene activity is presented as light units per mg of proteins.
Error bars represent standard deviation (n = 5)
we do not know the detailed mechanism how the
present nanoparticles were transported, furthermore
study is needed to confirm. Whatever the mechanism
was, because the present nanoparticle was only
10*12 nm in diameter and sufficient positeve zeta
pontential, which should play an important role in
DNA transport. Furthermore, because of its easy
availability, cheep source; simple preparation method
and excellent biocompatibility of the Nac-6-Au
nanoparticles thus will be more attractive vector for
gene delivery, especially oral gene therapy.
Acknowledgement This work was supported by the
Regional Research Centers Program of the Korean Ministry
of Educational and Human Resources Development through
the center for Healthcare Technology Development.
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