RESEARCH PAPER

Hydrophobically modified chitosan/gold nanoparticles forDNA delivery

Shanta Raj Bhattarai Æ Remant Bahadur K.C. ÆSantosh Aryal Æ Narayan Bhattarai ÆSun Young Kim Æ Ho Keun Yi Æ Pyoung Han Hwang ÆHak 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

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

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

123

J Nanopart Res (2008) 10:151–162

DOI 10.1007/s11051-007-9233-7

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

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

152 J Nanopart Res (2008) 10:151–162

123

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

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

Fig. 1 Chemical structure

chitosan. Subscripts m and

n represent the variable

number 78 and 22

respectively

J Nanopart Res (2008) 10:151–162 153

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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-

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.

154 J Nanopart Res (2008) 10:151–162

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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 48Cfor 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

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

J Nanopart Res (2008) 10:151–162 155

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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).

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

Fig. 2 UV absorbance spectra of; gold hydrosol (A), N-acylated chitosan-gold (Nac-6-Au) (B)

156 J Nanopart Res (2008) 10:151–162

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decreased to + 20 mV at pH 7.4 as complexes with

plasmid DNA. Nac-6-Au nanoparticles with or with-

out DNA results that the addition of plasmid DNA

increased the hydrodynamic diameter (13.33%) of the

nanoparticles. Furthermore, it can be inferred that the

f-potential of Nac-6, Nac-6-Au and Nac-6-Au/DNA

depends upon pH of resulting solution, and markedly

reduces (55.5*66.6%) after addition of the plasmid

DNA at pH (10 and 7.4) (Table 1). However, the

f-potential of Nac-6-Au with the plasmid DNA at

physiological condition (pH 7.4) is still acceptable for

transfection of mammalian cells.

Evaluation of cytotoxicity

Cytotoxicity of gene transfection vectors including

viral vectors, cationic liposomes and polymeric

cations is a major barrier to efficient delivery of

exogenous genes. Whether the presently formulated

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-

toxicities from three different experiments with

increasing concentration of the Nac-6-Au nanoparti-

cles. The Nac-6-Au nanoparticles at low concentration

(<16 mg/ml) showed relatively no significant toxicity

on the cells. The cell viabilities in the presence of

Nac-6-Au nanoparticles suspension ranged between

98% and 110% of the control in all experiments. At a

maximum Nac-6-Au nanoparticles concentration

(>32 mg/ml), the mean cell viabilities of the three

different cell lines showed about 89–96% viability

compared with that of the control. Interestingly, even at

Fig. 3 Size and size distribution of nanoparticles; Nac-6-Au

(a, A) and Nac-6-Au/DNA (a, B). Size was measured

using photon correlation spectroscopy (dynamic light scatter-

ing, DLS) and data were plotted as number distribution.

Transmission electron micrograph (TEM) of Nac-6-Au (b,

A), Nac-6-Au/DNA (b, B), and Nac-6-Au with higher

concentration of plasmid DNA (b, C). Scale bar represents

30 nm

Table 1 Variation in f-potential (mV) of nanoparticles at

different composition

Samples f -potential (mV)

(pH 10)

f -potential (mV)

(pH 7.4)

Nac-6a +50 +55

Nac-6-Aub +30 +40

Nac-6-Au/DNAc +10 +20

a N-acylated chitosanb N-acylated chitosan/goldc N-acylated chitosan/gold/DNA

J Nanopart Res (2008) 10:151–162 157

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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

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 concentrat ion of the part icles

(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

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-

sent standard deviation (n = 3). Control means the cells

growing in normal condition without adding the Nac-6-Au

nanoparticles

158 J Nanopart Res (2008) 10:151–162

123

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

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

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

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

J Nanopart Res (2008) 10:151–162 159

123

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

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

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)

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

160 J Nanopart Res (2008) 10:151–162

123

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

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.

References

Artursson P, Lindmark T, Davis SS, Illum L (1994) Effect of

chitosan on the permeability of monolayers of intestinal

epithelial cells (Caco-2). Pharm Res 11:1358–1361

Ana Y, Zhaia P, Dashtia AM, Wua S, Lina X, Wub MA (2002)

Combined gene delivery by co-transduction of adenoviral

and retroviral vectors for cancer gene therapy. Cancer Lett

184:179–188

Aral C, Akbuga J (1999) Preparation and in vitro transfection

efficiency of chitosan microspheres containing plasmid

DNA: poly (l-lysine) complexes. J Pharm Sci 6:73–82

Aryal S, KC RB, Dharmaraj N, Bhattarai N, Kim CH, Kim HY

(2006) Spectroscopic identification of S–Au interacton in

cysteine capped gold nanoparticles. Spectrochimica Acta

Part A 63:160–163

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

gene activity is presented as light units per mg of proteins.

Error bars represent standard deviation (n = 5)

J Nanopart Res (2008) 10:151–162 161

123

Bhattarai N, Bhattarai SR, Yi HK, Lee JC, Khil MS, Hwang

PH, Kim HY (2003) Novel polymeric micelles of

amphiphilic triblock copolymer poly (p-Dioxanone-co-l-

Lactide)-block-Poly (ethylene glycol). Pharm Res 20:

2021–2027

Bhattarai SR, Yi HK, Bhattarai N, Hwang PH, Kim HY (2006)

Novel block copolymer (PPDO/PLLA-b-PEG): Enhance-

ment of DNA uptake and cell transfection. Acta Bioma-

terialia 2:207–212

Chae SY, Son S, Lee M, Jang MK, Nah JW (2005) Deoxy-

cholic acid-conjugated chitosan oligosaccharide nanopar-

ticles for efficient gene carrier. J Control Release

109:330–334

Corsi K, Chellat F, Yahia H, Fernades JC (2003) Mesenchymal

stem cells, MG63 and HEK293 Transfection using

chitosan-DNA nanoparticles. Biomaterials 24:1255–1264

Chakrabarti R, Klibanov AM (2003) Nanocrystals modified

with peptide nucleic acids (PNAs) for selective self-

assembly and DNA detection. J Am Chem Soc 125:

12531–12540

Daniel MC, Astruc D (2004) Gold nanoparticles: assembly,

supramolecular chemistry, quantum-size-related proper-

ties. Chem Rev 104:293–346

Ferrari S, Duncan GM, Alton WFW (2002) Barriers to and new

approaches for gene therapy and gene delivery in cystic

fibrosis. Adv Drug Delivery Rev 54:1373–1393

Iqbal M, Lin W, Jabbal-Gill I, Davis SS, Steward MW, Illum L

(2003) Nasal delivery of chitosan–DNA plasmid

expressing epitopes of respiratory syncytial virus (RSV)

induces protective CTL responses in BALB/c mice.

Vaccine 21:1478–1485

Ishii T, Okahata Y, Sato T (2001) Mechanism of cell transfection

with plasmid/chitosan complexes. BBA-Biomembranes

1514:51–64

Kai E, Ochiya T (2004) A method for oral DNA delivery with

N-Acetylated Chitosan. Pharm Res 21:838–843

KC RB, Aryal S, Bhattarai SR, Bhattarai N, Kim CH, Kim HY

(2006) Stabilization of gold nanoparticles by hydropho-

bically modified polycations. J Biomater Sci Polym Ed

17(5):576–589

Kim TH, Park IK, Nah JW, Choi YJ, Cho CS (2004) Galac-

tosylated chitosan/DNA nanoparticles prepared using

water-soluble chitosan as a gene carrier. Biomaterials

25:3783–3792

Koping-Hoggard M, Guan IT, Edwards K, Nilsson M, Varum

KM, Artursson P (2001) Chitosan as a nonviral gene

delivery system. Structure–property relationships and

characteristics compared with polyethylenimine in vitro

and after lung administration in vivo. Gene Therapy 8:

1108–1121

Kulmeet IS, Catherine MM, Joseph MS, Sallie WS, Vincent

MR (2002) Gold nanoparticles mediated transfection of

mammalian cells. Bioconjugate Chememistry 13:3–6

Lee KY, Kwon IC, Kim YH, Jo WH, Jeong SY (1998) Rep-

aration of chitosan self-aggregates as a gene delivery

system. J Control Release 51:213–220

Leong KW, Mao HQ, Truong-Le VL, Roy K, Walsh SM,

August JT (1998) DNA-polycation nanospheres as non-

viral gene delivery vehicles. J Control Release 53:

183–193

Lundstrom K (2003) Latest development in viral vectors for

gene therapy. Trends Biotechnol 21:117–122

Le Tien C, Lacroix M, Ispas-Szabo P, Mateescu MA (2003) N-

acylated chitosan: hydrophobic matrices for controlled

drug release. J Control Release 93:1–13

Maclaughlin FC, Mumper RJ, Wang J et al (1998) Chitosan

and de-polymerized chitosan oligomers as condensing

carriers for in vivo plasmid deliver. J Control Release

56:259–272

Mao HQ, Roy K, Troung-Le VL, Janes KA et al (2001)

Chitosan-DNA nanoparticles as gene carriers: synthesis,

characterization and Transfection efficiency. J Control

Release 70:399–421

Park IK, Kim TH, Park YH, Shin BA, Choi ES et al (2001)

Galactosylated chitosan-graft-poly (ethylene glycol) as

hepatocyte-targeting DNA carrier. J Control Release 76:

349–362

Pertmer TM, Eisenbraun DM, McCabe D, Prayaga SK, Haynes

JR (1995) Gene gun-based nucleic acid immunization:

elicitation of humoral and cytotoxic T lymphocyte re-

sponses following epidermal delivery of nanogram

quantities of DNA. Vaccine 13:1427–1430

Quong D, Yeo JN, Neufeld RJ (1999) Stability of chitosan and

poly-l-lysine membrane coating DNA-alginate beads

when exposed to hydrolytic enzymes. J Microencapsula-

tion 16:73–82

Quong D, Neufeld R (1998) DNA protection from extracap-

sular nucleases, within chitosan or poly-l-lysine-coated

alginate beads. J Biotechnol Bioeng 60:124–134

Ravi Kumar MNV, Bakowsky U, Lehr CM (2004) Preparation

and characterization of cationic PLGA nanospheres as

DNA carriers. Biomaterials 25:1771–1777

Roy K, Mao HQ, Huang SK, Leong KW (1999) Oral gene

delivery with chitosan-DNA nanoparticles generates

immunologic protection in a murine model of peanut al-

lergy. Nature Med 5:387–391

Ruponena M, Honkakoskia P, Ronkko S, Pelkonenb J, Tammic

M, Urtti A (2003) Extracellular and intracellular barriers

in non-viral gene delivery. J Control Release 93:213–217

Schuber F, Kichler A, Boeckler C, Frisch B (1998) Liposomes:

from membrane models to gene therapy. Pure Appl Chem

70:89–96

Thanou M, Florea BI, Geldof M, Junginger HE, Borchard G

(2002) Quaternized chitosan oligomers as novel gene

delivery vectors in epithelial cell lines. Biomaterials 23:

153–159

Tripathy SK, Black HB, Goldwasser E, Leiden JM (1996)

Immune responses to transgene-encoded proteins limit the

stability of gene expression after injection of replication-

defective adenovirus vectors. Nat Med 2:545–550

162 J Nanopart Res (2008) 10:151–162

123