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 Ó Springer Science+Business Media B.V. 2007

Abstract Present study dealt an application of modified chitosan gold nanoparticles (Nac-6-Au) for the immobilization of necked plasmid DNA. Gold nanoparticles stabilized with N-acylated chitosan were prepared by graft-onto approach. The stabilized gold nanoparticles were characterized by different physicochemical 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 Á Remant Bahadur K.C. Á 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 Á 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-dispersed 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 nanoparticles. 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 Á DNA delivery Á Gene therapy Á Gold nanoparticles Á Non viral vectors Á Nanomedicine

Introduction Gene therapy holds an excellent means for curing acquired and inherited diseases in a straightforward

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1994). However. 2004. synthetic polymers etc (Schuber et al. 2002). 2003). Ruponena et al. the protonated amino groups of chitosan and chitosan-based materials can effectively bind to DNA and condense it as nano/microparticles (Lee et al. Generally. 1998. Chitosan also increases the transcellular and paracellular transport across mucosal epithelium (Artursson et al. Many attempts have been performed for the betterment of gene delivery using non-viral vectors viz: biomolecules. 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. have been employed as an effective carrier due to their excellent electrostatic interaction with therapeutic genes (KopingHoggard et al. 1995). The need of current methodology is to attribute these limitations (Tripathy et al. Preclinical 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. poly-l-lysine (Aral and Akbuga 1999. Ferrari et al. 1996). In recent years. modification (chemical and physical) of natural chitosan is supposed to be an excellent means for the formulation of better gene delivery vehicle. Iqbal et al. and oral administration has been largely ignored due to the large hurdles that need to be overcome for gene delivery. So. potentiality of chitosan as a nonviral gene carrier has been extensively considered (Roy et al. Quong and Neufeld 1998) have been frequently performed for the formulation of effective chitosan and chitosan-based materials to enhance the efficiency of gene delivery. the use of chitosan and chitosanbased materials as a gene carrier remains inadequate due to uncontrolled size and inappropriate processing media (insoluble in physiological pH). Thanou et al. Moreover. targeting efficiency etc in their in vivo and in vitro use. the use of gold as gene carrier in an aqueous medium has several limitations because of its rapid aggregation. 2004). 2001. 2005. natural polymers. non-viral mediated systems have attracted a great deal of interest in this field. Ana et al. Ishii et al. Non-viral mediated gene transfer vehicles with appropriate functional groups. 2001. Various approaches viz: modification with ligands (Mao et al. 2002). Two major delivery systems have been used in the current gene therapeutic approaches viz: viral and non-viral mediated system (Lundstrom 2003. 2003). 1998. despite their comparatively low efficiency. in vitro and in vivo is the major limitation of nonviral mediated gene therapeutic approaches (Tripathy et al. 2002). such use is not so frequent due to some sever limitations like: restricted immunogenicity. The beauty of our formulation was significant stability of N-acylated chitosan stabilized gold nanoparticles in physiological condition. On the other hand. In acidic pH. On the other hand. 1998. hydrophobically modified chitosan has also been used in gene delivery (Chae et al. the N-acylated chitosan play dual roles as a stabilizer and a carrier. lipases and peptidases present in the gastrointestinal tract. 1999). 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. blending with polymers. we already explored formulation procedure of chitosan and gold so as to overcome their limitation. 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. and poor permeability of both genes and gene vectors across the intestinal epithelium owing to the size and charge of gene delivery vehicles. Realizing their potential application in gene delivery. gold particles 123 . further indicative of its potential in oral gene delivery and in generating protective mucosal immune responses. 2001. Maclaughlin et al. Leong et al. 1998. However. most gene delivery strategies have focused on the parenteral route of delivery. However. correcting. 2003. Inorganic nanoparticles (silica or gold) is an inert materials with no obvious sensitivity with acid pH and intestinal digestive enzymes. 2004). Park et al. 2002. Kai et al. and replacing the affected genes. Efficient delivery of therapeutic genes into the target cells. Ravi Kumar et al. Quong et al. Here.152 J Nanopart Res (2008) 10:151–162 way by adding. 1999). and chitosan is a natural biodegradable and biocompatible mucoadhesive polysaccharide that has been widely used in oral gene delivery (Roy et al. Hence. which are protonated at physiological pH. Mao et al. pathogenicity. Recently. 2001). Kim et al. 1999. 1996). 2001. Viral-mediated systems are the most effective means for delivery and expression of gene. such as acidity in stomach. the nuclease.

1 · 105. the preparation of N-acylated chitosan (Nac) was done using different fatty acyl chlorides (Le et al. However. sodium borohydride were purchased from Sigma-Aldrich Co. All other chemicals were purchased from Showa chemical Ltd. Each measurement Fig. f-potential of the nanoparticles was determined by electrophoratic light scattering (ELS) (ELS 8000/ 6000 Otsuka electronics Co. Ltd. and used without any further purification. Hydrophobic modification of native chitosan i. Preparation of self-assembled N-acylated chitosan/gold (Nac-6-Au) nanoparticles Chemical structure of native and N-acylated chitosan is shown in Fig. hexanoyl chloride and octanoyl chloride). Reagents Chitosan-10 (viscosity average molecular weight. freshly prepared 123 .e. Subscripts m and n represent the variable number 78 and 22 respectively was performed at room temperature after sonicating the samples into an ultra-sonicator bath for 1 min. hydrogen tetrachloroaurate (HAuCl4). Viscosity average molecular weight of chitosan was determined according to the previous report (KC et al.. Experimentals Instrumental UV-vis absorption spectra of the samples were recorded in Cary 500 UV-vis-NIR spectrometer. Samples for TEM were prepared by dipping a carbon-coated copper grid in an aqueous dispersion of nanoparticles and dried at room temperature. 1. degree of deacetylation 78%) was purchased from Wako Pure Chemical Industries.. Japan) with measuring angle of 208 to incident beam. 1 Chemical structure chitosan. Particle size and morphology were observed by JEOL JEM 2010 transmission electron microscope (TEM) operating at 200 kV. monodisperse nanoparticles. 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 (Cyonics) with measuring angle of 908 to the incident beam. in-vitro and in-vivo. Mv = 2. 2003). Grafting of Nac on gold nanoparticles (Nac-Au) was taken from previous publication (KC et al. and act as contrast agent while detecting delivery site. Japan.. 2006).J Nanopart Res (2008) 10:151–162 153 provide the nanoscopic. 2006). Fatty acyl chlorides (e.. of Korea. Briefly. current study describes hydrophobically modified chitosan stabilized gold nanoparticles as a novel DNA carrier for gene delivery.g.

1His/Myc/ LacZ plasmid was used for preparation of complexes in phosphate buffer (PBS. The resulting mixture was stored for 30 min at room temperature and then used in DNA uptake or transfection experiment.0 ml polymer solution (33% in 0. Nac-8-Au) have been formulated. and grown at 378C under 5% CO2 atmosphere as described in our previous report (Bhattarai et al. The cytotoxicity of Nac/Au nanoparticles was evaluated in comparison with control cells. out of which Nac-6-Au was selected for the DNA delivery due to its higher stability (KC et al. MTT-containing medium was aspirated off and 150 ll of DMSO were added to dissolve the crystals formed by living cells. The following media were used: 1. RPMI-1640 medium containing with 10% (v/v) fetal bovine serum (FBS) (Gibco) for 3T3 cells. Cells were incubated for additional 24 h after the addition of defined concentration of Nac/Au nanoparticles. penicillin (100 U/ml) and streptomycin (100 lg/ml) was used. Plasmid amplification The procedure for plasmid amplification was taken from our previously published report (Bhattarai et al. Preparation of DNA complexes Nac-6-Au nanoparticles and pcDNA3.0 ml) was added to the 2.4). mouse embryo cell. CT-26. During transfection experiment cells were supplemented with Nac-6- Au/DNA complexes. pH 7. freshly prepared ice-cold sodium borohydride (0. Purity was conformed by 1% Agarose gel electrophoresis followed by Ethdium bromide (EtBr) staining. plasmid DNA (pcDNA3. breast cancer cell) were used for transient transfection experiments and cytotoxicity. and incubated for 4 h at 378C. using a microplate reader (ELX 800. 2003). BIOTEK Instruments. 123 . containing 10% FBS were distributed in a 96-well plates. Inc.154 J Nanopart Res (2008) 10:151–162 HAuCl4 aqueous solution (10 mM. 2003). and 2. Briefly. and again incubated in same condition up to 48 h. and DNA concentration was measured by UV absorption at 260 nm. 20 ll of MTT solution (5 mg/ml in 1 · PBS) were added to each well. 2006). Evaluation of cytotoxicity Evaluation of the cytotoxicity was performed by the MTT assay in four kinds of cell lines (MCF-7. Briefly. Here two types of Nac-Au (Nac-6-Au. After 4 h. and the plates were slowly agitated for 2 min. and incubated in a humidified atmosphere containing 5% CO2 at 378C for 24 h (Bhattarai et al. Rapid color change to pink indicates the formation of gold nanoparticles. and quantified by b-galactosidase assay. Next. 1. Results were observed by X-gal staining method. Cell line preparation Cells (NIH 3T3. For all media. The plasmid DNA was purified by phenol–chloroform and was diluted in sterilized water.6 kb containing bacterial b-galatosidase gene (LacZ with a size of 1. media was replaced by fresh media containing 10% FBS. To this solution.1 M. Thus formed gold nanoparticles were purified and collected using ultracentrifuge operated at 35. Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) with 10% (v/v) fetal calf serum (Gibco) for CT-26 and MCF-7 cells. 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.4 ml) was added under moderate stirring at room temperature. The cell viability (%) was calculated according to the following equation: Cell viability (%) = [OD490(sample)/OD490(control)] · 100.).2 kb) as the reporter gene under the control of CMV (cytomegalovirus) promoter was used in this study. The mixture was replaced with fresh medium containing 10% FBS.1His/Myc/ LacZ) (Invitrogen.000 g for 30 min at 48C. 0. The transformed cells were grown in large quantities of LB broth supplemented with Ampicillin (10 mg/ml). USA) with a size of 6.1 M HCl) and stirred for 1 h. 2006). The plate was incubated for an additional 4 h at 378C. coli) JM109 Bacterial strain was used as host cell for amplification of plasmids. Absorbance was measured at 490 nm. Escherichia coil (E. Then. 3T3 and CT-26 cells). colon cancer cell and MCF-7. 5% CO2 atmosphere. various cell suspensions containing 1 · 104 cell/well in RPMI-1640 for NIH3T3 cell and DMEM for MCF-7 and CT-26.

1 mM MgSO4. 50 mg per mice) or plasmid DNA (pcDNA-LacZ) with Lipofectin. 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. Briefly. Culture media were changed with fresh complete media containing defined concentration of Nac-6-Au/DNA nanoparticles as described in preparation of DNA Complexes. 10 mM KCl. Lipofectin1 (Invitrogen. For the comparison purpose commercially available transfection kit. 2 mM K3Fe (CN)6. cells were harvested for b-galactosidase assay. with their stomachs and small intestines harvested and homogenized for 20 s with 1 ml lysis buffer containing 123 . 0. Protein concentration of cell lysate was determined with Bradford method.J Nanopart Res (2008) 10:151–162 155 Analysis of DNA complexes DNA complexes corresponds to the DNA binding with the nanoparticles was first analyzed by spectrophotometer. The b-galactosidase assay was performed in a microtiter dish. the mice were killed and their stomachs and small intestines were surgically removed. A galacto-Star Kit (Tropix. suspended in PBS.4) was added to the reaction mixture and incubated for 1*16 h at 378C. Then. and 50 mM 2-mercaptoethanol. MA. and furthermore verified with gel electrophoresis. USA) was also used during the transfection study. 50 ll ONPG (O-nitrophenyl-b-d-galactopyranoside) substrate solution (4 mg/ml ONPG in 100 mM phosphate buffer. and units of enzyme were expressed as nanomoles of b-galactose formed per min. The C57BL/6 mice were fed either Nac-6-Au nanoparticles containing the LacZ gene (pcDNA-LacZ. All mice were used in experiment at 7–8 weeks of age. 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). Chuungnam. Bedford. and incubated for 5 min at 378C. 10 · PBS) for overnight at 378C. and 1 mM DTT by freezing and thawing.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 spectrophotometer analysis at OD = 260.2% Triton X-100. After 48 h of incubation. Resulting samples were stored in room temperature for 6 h and then centrifuge at 13. 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. pH 7.4). About 25 ll of cell lysate was added to 135 ll of buffer containing 100 mM KH2PO4/K2HPO4 (pH 7. The mice were maintained in animal facilities at the Chonbuk National University and used in accordance with the guidelines of the University. Three days later. 2 mM K4Fe (CN)6. mice were sacrificed. at defined times after oral delivery.4). The assay was done as previously described method (Bhattarai et al. The cells were lysed in 200 ll of lysis buffer containing 100 mM KH2PO4/ K2HPO4 (pH 7. USA) was used to measure in vivo reporter gene expression. 2 mM Mgcl2. b-galactosidase activity (U/mg of total protein in lysate) = [OD 420/0. 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. using animal feeding needles. After fixing 1 h. Transfection of cells corresponding to the expression of blue color was monitored by light microscope and images were digitally photographed. The bgalactosidase activity was calculated by using the following equations. 0. and collected by centrifugation. Remaining supernatant portion was discarded. In vivo gene expression Female C57BL/6 mice were purchased from the Korean Research Institute of Chemical Technology (Daejeon. After the incubation period. The cells were detached with trypsin. 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. Briefly. Korea) and were housed in an environment-controlled rearing system. Samples were prepared as described in preparation of DNA complexes. 2006).0045 · assay volume (ml)] minÀ1 mgÀ1. culture media were discarded and the cells were washed with PBS.2% (v/v) glutaraldehyde 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.

A. TEM micrograph of Nac-6-Au nanoparticles showed a well dispersed. C) moreover aggregated. N-acylated chitosan-gold (Nac-6-Au) (B) 123 .4 after incorporation of gold. USA) and incubated at room temperature for 60 min. Two hundred micrograms of protein from each sample was mixed with 70 ll reaction buffer in Monolight Luminometer cuvettes (Pharmingen. Germany) and centrifuged at 12. On partial acylation. The nanoparticles of Nac-6 being a polycation gives different +ve f–potential depending on the pH of media. B). 3b. 2006). The sample was centrifuged again and measured total protein concentration. gold hydrosol (A). spherical and regular nanoparticle with average size 12. which is one of the hindering factor in gene delivery. 3a.156 J Nanopart Res (2008) 10:151–162 protease inhibitors cocktail (Boehringer Mannhein. Fig. and further 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 plasmon band (SPB) at 512. 3b. B). 2). Table 1 shows the f-potential of Nac-6. However.9 ± 0. The average size of Nac-6-Au nanoparticles without DNA was 13. The f-potential of the Nac-6 was increased up to + 40 mV at pH 7. Chakrabarti and Klibanov 2003. 2006).34 nm (Fig. highly disperse and suitable for mammalian cells uptake (Fig. suggesting the formation of gold nanoparticles (Fig. the nanoparticles with plasmid DNA (at low concentration) observed in this study were relatively small. 3b. San Diego. Aryal et al.5 nm where as with DNA was 15. The supernatant fluid was heated at 488C for 60 min to inactivate endogenous b-galactosidase activity. The result of DLS measurement showed a uni-model size distribution of nanoparticles without DNA and with DNA. Nac-6-Au and Nac-6-Au/DNA. 3b A).4. but with the increase in the concentration of DNA the shape of individual nanoparticles was clustered (Fig. Furthermore characterization of the particles was taken from the previous publication (KC et al. CA. respectively. 2 UV absorbance spectra of. The bgalactosidase activity is expressed as relative light units per milligram protein (U/mg). B). The shape and regularity of nanoparticles with DNA at low concentration was not so different from Nac-6Au nanoparticles (Fig. and 541 nm.2 nm (Fig. f-potential of the Nac-6 was + 50 mV at pH 10 where as that potential was + 55 mV at pH 7. 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. 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.500 g for 10 min at 48C.

even at Table 1 Variation in f-potential (mV) of nanoparticles at different composition Samples Nac-6a Nac-6-Aub Nac-6-Au/DNA a b c c f -potential (mV) (pH 10) +50 +30 +10 f -potential (mV) (pH 7. it can be inferred that the f-potential of Nac-6.33%) of the nanoparticles. Interestingly. Nac-6-Au/DNA (b. Whether the presently formulated vector (Nac-6-Au nanoparticles) influenced cell viability was investigated in three different cell lines.4 as complexes with plasmid DNA.4) is still acceptable for transfection of mammalian cells. However. Nac-6-Au and Nac-6-Au/DNA depends upon pH of resulting solution. and markedly reduces (55. Scale bar represents 30 nm decreased to + 20 mV at pH 7. B). Nac-6-Au (a. Figure 4 shows the representative data of cytotoxicities from three different experiments with increasing concentration of the Nac-6-Au nanoparticles. cationic liposomes and polymeric cations is a major barrier to efficient delivery of exogenous genes.5*66. Evaluation of cytotoxicity Cytotoxicity of gene transfection vectors including viral vectors.4) (Table 1). DLS) and data were plotted as number distribution. A) and Nac-6-Au/DNA (a. the f-potential of Nac-6-Au with the plasmid DNA at physiological condition (pH 7. Nac-6-Au nanoparticles with or without DNA results that the addition of plasmid DNA increased the hydrodynamic diameter (13. 3 Size and size distribution of nanoparticles.6%) after addition of the plasmid DNA at pH (10 and 7. and Nac-6-Au with higher concentration of plasmid DNA (b.J Nanopart Res (2008) 10:151–162 157 Fig. MTT assays were performed to evaluate the cytotoxicity. Size was measured using photon correlation spectroscopy (dynamic light scattering.4) +55 +40 +20 N-acylated chitosan N-acylated chitosan/gold N-acylated chitosan/gold/DNA 123 . the mean cell viabilities of the three different cell lines showed about 89–96% viability compared with that of the control. C). Furthermore. Transmission electron micrograph (TEM) of Nac-6-Au (b. A). At a maximum Nac-6-Au nanoparticles concentration (>32 mg/ml). The cell viabilities in the presence of Nac-6-Au nanoparticles suspension ranged between 98% and 110% of the control in all experiments. B). The Nac-6-Au nanoparticles at low concentration (<16 mg/ml) showed relatively no significant toxicity on the cells.

the bright band of DNA was not significant. 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. However.0*6.158 J Nanopart Res (2008) 10:151–162 high concentrations of Nac-6-Au nanoparticles up to 45 mg/ml.5 mg/ml) of the Nac-6-Au nanoparticles was not destructive for plasmid DNA. it was shown that the Nac-6-Au nanoparticles suspension was not toxic to the cell at low concentration. 4 Cell viability assay.0 mg/ml).5 mg/ml). In our separate experiment. which is 10*15-fold higher than the concentration required for high efficiency of transfection. at the higher concentration. respectively. The cell viability was estimated after 36 h using MTT colorimetric assay. and accumulate around the cell membrane. Amount of DNA in gel was significantly changed after adding different concentration (1.5 mg/ml of the particle concentration. and interfere the normal biological process. Nac-6-Au showed no obvious negative effect on cell viability. Results were analyzed on the basis of observation by comparing the brightness of DNA bands Fig. In contrast. the absorbance was significantly decreased and was minimum at 4. higher concentration (>16 mg/ml) of the Nac-6Au nanoparticles may aggregate. Figure 5 shows bar diagram and gel electrophoresis to determine complex forming capacity corresponds to the DNA binding with Nac-6-Au nanoparticles. 5 (Lane 8). Error bars repre- sent standard deviation (n = 3). It means that the DNA did not interact with the Nac-6-Au nanoparticles. 5 (Lanes. From cytotoxicity results. From these two results (spectrophotometer and gel electrophoresis).0*2. 4. Fig. The assays performed in triplicate and standard error is shown.0*4.5 mg/ml Nac-6-Au nanoparticles. Fig. It Fig. 1 to 2). Most of the polycations can bind to the negatively charged plasma membrane and destabilize them. results from the gel electrophoresis showed that even higher concentration ( >4. At the lower concentration of the nanoparticles (1.0 mg/ml. Furthermore.0 mg/ml to 6. the reduction in membrane toxicity in present study could be due to well dispersability of Nac-6-Au nanoparticles in aqueous medium consequently suppress the interaction with cell membrane But. we concluded that the Nac6-Au nanoparticles at optimum concentration (4. Moreover. Decreased absorbance means the decreased plasmid DNA in supernatant corresponds to the binding or complexes with the nanoparticles and settles down as sediment.0 mg/ml) of the Nac-6-Au particles from the stock 50 mg/ml. Analysis of DNA complexes Complex formation between plasmid DNA and the Nac-6-Au nanoparticles is correlated with DNA binding with the nanoparticles. it has been investigated that the cytotoxicity correlates with membrane damage effect. these results were verified by sedimentation analysis using gel electrophoresis. 5 (Lanes 3 to 8) and highly bright (high concentration of DNA) at the concentration of 4. But this band was significantly increased with increasing concentration of the particles (2. which may lead the cytotoxic effect. Fig. Bar diagram represents the results of spectrophotometer with increasing concentration of the Nac-6-Au nanoparticles from 1. Control means the cells growing in normal condition without adding the Nac-6-Au nanoparticles 123 .5 mg/ml) could have complex forming capacity with the DNA.

5. 2. Furthermore. this paper studies the complexes having the optional composition of the nanoparticles (4.5 mg/ ml) of the nanoparticles. 4. From each sample.0. 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).5 mg/ ml) was shown to be most effective for transfection on three different cell line (3T3. 5. Marker means Hind III and control means pure plasmid DNA with out Nac-6-Au nanoparticles.0 mg/ml to 6. That’s why. 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. 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/nanoparticles complexes as shown in Fig.5.5.000 g/48C) for 15 min.0 and 5. However. CT 26 and MCF-7). 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. the present nanoparticles may bind to cells via their net positive 123 .0* 6.0 mg/ml was added into the constant amount of the plasmid DNA (5 lg).0. data shown here is only one cell line (MCF-7) because of its higher b-galactosidase activity compared to the other cell lines (3T3 and CT 26). 2. all samples were run on a 1% agarose gel and stained with ethidium bromide (EtBr).5. Over all visual bands also indicate that there was not destructive interaction between Nac-6-Au nanoparticles and plasmid DNA means. supernatant solution was used for spectrophotometer analysis and sediment part was used for gel electrophoresis. Error bars represent standard deviation (n = 3). Transfection on MCF-7 cells is our promising result. 4.0. so far we are unable to predict the mechanism of action of the present nanoparticles. 3. Furthermore. Lanes (1*2) shows almost lack of plasmid DNA where as lane (8) shows maximum plasmid DNA. which was significantly increased when the plasmid DNA mixed with different concentration of the N-acylated chitosan gold (Nac-6-Au) nanoparticles (1. Different concentration of the Nac6-Au nanoparticles from 1. which remains to be further explored. High internalization (plasmid DNA uptake) corresponds to the higher value of b-galactosidase activity. 1.0. there may be some possibility that present nanoparticles may probably involve an important role.J Nanopart Res (2008) 10:151–162 159 Fig. However. 3. the present nanoparticles may increase the bioavilability of plasmid DNA for in vivo application. Lanes (1*10) contain the Nac-6-Au nanoparticles concentration 1.0 mg/ml) on MCF-7 cell.5 mg/ml with constant amount of the plasmid DNA (5 lg). At optimum concentration (4. For gel electrophoresis. it has been suggested that the efficacy of transfection with complexes formed between DNA and cationic polymers strongly depends upon the complex composition. 5 (Lane 8). The resulting solution was vortex for 10 min and kept for 6 h at room temperature before centrifuge (13. The presence and absence of serum in the transfection medium did not affect the transfection efficiency (data not shown).

Although naive mice and mice fed Lipofectin/DNA showed some activity. The reason behind it would be inorganic nanoparticles (gold) is an inert materials with no obvious sensitivity with acid pH and intestinal digestive enzymes. To assess the expression and distribution of transduced genes after oral DNA delivery. on average. 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). Error bars represent standard deviation (n = 3). b-gal reporter gene activity is presented as light units per mg of proteins. A and B). in vitro. we fed C57BL/6 mice either Nac-6-Au/DNA nanoparticles containing the LacZ gene or plasmid DNA (LacZ) with Lipofectin1. specifically in the transfection medium. Higher transfection efficiency corresponds to the number of blue colored cell was clearly seen in both photographs. However. 7). Based on this hypothesis.0 mg/ml) of the Nac-6-Au nanoparticles with constant amount of plasmid DNA (5 lg). The activity sections represent. mice fed the Nac-6-Au/DNA nanoparticles showed a higher level of gene expression in both the stomach and small intestine.5 mg/ml concentration with the commercial transfection reagents (Lipofectin1. b-galactosidase activity was 15*20 times higher expression with Nac-6-Au/DNA nanoparticles compared to the Lipofectin method. furthermore conditions should be optimized.160 J Nanopart Res (2008) 10:151–162 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. we compared the transfection efficiency of Nac-6-Au nanoparticles at 4. pH of media solution etc.0*6. Interestingly. 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 123 . 6 can be interpreted. and Fig. an increased transfection efficiency of Fig. presently formulated Nac-6Au/DNA system seems to be highly applicable in invivo especially to oral gene delivery. Fig. Furthermore. 6 (photographs. 50% of the whole small intestine. 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). 1 mg/ml) on MCF-7 breast cancer cells. the present Nac-6-Au nanoparticles showed suitable carrier for gene delivery. In contrast to in-vitro. Our studies did not optimize different conditions like time courses for maximum gene expression. 6 Bar diagram represents the transfection efficacy using b-galactosidase assay on MCF-7 cells with different concentrations (1. in limited condition. We determined the tissue expression of bacterial b-galacotosidase (LacZ) in the stomach and small intestine 3 days after the oral administration (Fig. We further compared this activity and found highly expression in intestine compared to stomach. For its better use.

and considerable size and fpotential in physiological (pH 7. KC RB. Wua S. At present we do not know the detailed mechanism how the present nanoparticles were transported.4) for DNA delivery. Furthermore. Kim CH. Davis SS. Whatever the mechanism was. Chitosan also increases the transcellular and paracellular transport across mucosal epithelium (Artursson et al. Lina X. Error bars represent standard deviation (n = 5) chitosan is a natural biodegradable and biocompatible mucoadhesive polysaccharide. because of its easy availability. 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. 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. furthermore study is needed to confirm. Akbuga J (1999) Preparation and in vitro transfection efficiency of chitosan microspheres containing plasmid DNA: poly (l-lysine) complexes. Pharm Res 11:1358–1361 Ana Y. Dharmaraj N. Kim HY (2006) Spectroscopic identification of S–Au interacton in cysteine capped gold nanoparticles. It was adopted by grafting N-acylated chitosan on the surface of gold nanoparticles that ensures the physico-chemical stability in aqueous medium at physiological pH 7. Bhattarai N. Above all characteristic feature suggest that chitosan or chitosna base stabilized gold nanoparticles could be a suitable vector for oral gene delivery. simple preparation method and excellent biocompatibility of the Nac-6-Au nanoparticles thus will be more attractive vector for gene delivery. Nac-6-Au/DNA nanoparticles complexes were prepared under defined conditions. Aqueous solution of the Nac-6-Au nanoparticles had ability to form complexes with plasmid DNA through electrostatic interaction. J Pharm Sci 6:73–82 Aryal S. b-gal reporter gene activity is presented as light units per mg of proteins. because the present nanoparticle was only 10*12 nm in diameter and sufficient positeve zeta pontential. Conclusion A stable and reproducible formulation of Nac-6-Au nanoparticles has been obtained via surface modification of gold nanoparticles. Lindmark T. cheep source. Cancer Lett 184:179–188 Aral C. 1994). Spectrochimica Acta Part A 63:160–163 123 . References Artursson P. Illum L (1994) Effect of chitosan on the permeability of monolayers of intestinal epithelial cells (Caco-2). Wub MA (2002) Combined gene delivery by co-transduction of adenoviral and retroviral vectors for cancer gene therapy. Moreover. Dashtia AM.4. especially oral gene therapy. The size of the Nacylated chitosan gold nanoparticles (Nac-6-Au) (after and before complex formation) was optimized to be in a nano-size range.J Nanopart Res (2008) 10:151–162 161 Fig. Zhaia P. further indicative of its potential in oral gene delivery and in generating protective mucosal immune responses. f-Potential of these particles/complexes was varied according to the pH. which should play an important role in DNA transport.

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