Targeted images of KB cells using folate-conjugated gold nanoparticles
© Pierson et al.; licensee Springer. 2015
Received: 26 November 2014
Accepted: 30 December 2014
Published: 21 January 2015
Mercaptosuccinic acid-coated gold (GM) nanoparticles were prepared and characterized by transmission electron microscopy and dynamic light scattering. Folic acid (F) was then conjugated to the GM to preferentially target oral squamous cancer (KB) cells with folate receptors expressed on their membranes and facilitate the transit of the nanoparticles across the cell membrane. Finally, a fluorescence dye (Atto) was conjugated to the nanoparticles to visualize their internalization into KB cells. After culture of the cells in a medium containing GM and folate-conjugated GM (GF), the interaction of surface-modified gold nanoparticles with KB cells was studied.
For more than one decade, nanometer-sized gold nanoparticles have attracted considerable attention not only because of their size- and shape-dependent optical and electronic properties that are distinctly different from the corresponding bulk materials but also due to their potential applications in thermal, catalysis, surface-enhanced Raman scattering, photoelectronic devices, biomedical diagnostics, and other related fields [1-3]. Gold nanoparticles offer important new possibilities in cancer diagnosis and therapy . They can be used for the location and visualization [5-7] of tumors in their primary and potentially also secondary locations, as delivery vehicles for anticancer drugs, and in non-invasive ablation therapies. Gold nanoparticles are also novel promising biocompatible nanoprobes, exhibiting surfaces and cores with specific physicochemical properties, e.g., optical chirality , fluorescence [9,10], near-infrared photoluminescence, , and ferromagnetism , which provide new opportunities for clinical diagnostics. The transport of the nanoparticles to the tumor is a multistage process . Systemically administered nanoparticles with tumor-binding ligands can accumulate in the tumors, owing to the more chaotic vasculature compared to non-diseased tissue .
As cancer remains extremely difficult to treat, effective strategies to detect it in its early stages are critical. In this respect, imaging has become an indispensable tool in clinical trials and medical practice . Fluorescent bio-imaging is also of great value for visualizing the expression and activity of particular molecules, cells, and biological processes that influence the behavior of tumors and/or their responsiveness to therapeutic drugs . Therefore, a wide range of fluorescent components has been explored by in vitro bio-imaging studies, including bio-marking of tumor tissues , angiogenic vasculature, and sentinel lymph nodes . In this respect, several kinds of nanomaterials such as quantum dots, noble metal nanoparticles, and new hybrid nanocomposites of reduced graphene oxide and gold nanoparticles have demonstrated great potential for highly sensitive optical imaging of cancer, on both cellular and animal levels.
The multifunctional properties of nanoparticles provide unique advantages for the cancer-specific delivery of imaging and therapeutic agents . Small molecules with infinitely diverse structures and properties are inexpensive to produce and have great potential as targeting moieties . Folate, one of the small molecules most widely studied as a targeting moiety, is a water-soluble vitamin B6, essential for rapid cell division and growth in humans, especially during embryonic development [7,20]. Li et al. designed folate receptor-targeted hollow gold nanospheres carrying siRNA recognizing NF-κB, a transcription factor related to the expression of genes involved in tumor development . Selim et al. demonstrated the use of folic acid-conjugated magnetites as a folate-targeted dual contrast agent for nuclear imaging .
In this study, we proposed the new gold nanoparticles with functions of molecular imaging and cell targeting. To produce hydrophilic and functional gold nanoparticles, mercaptosuccinic acid-coated gold (GM) nanoparticles were synthesized in an aqueous medium by the citrate reduction method at room temperature. Then, folic acid (F) was immobilized as a targeting moiety on the surface of the GM to ensure specific recognition of oral squamous cancer (KB) cells, to facilitate the uptake of nanoparticles into the cells, and to enhance the biocompatibility of the folate-conjugated GM (GF) nanoparticles. Animal fibroblast cells (NIH 3T3) were also used as a control. The surface properties of GM and GF were characterized by various physicochemical means, and the intracellular uptake of GF to the KB cells was also observed by confocal laser scanning microscope (CLSM).
Auric chloride (HAuCl4), mercaptosuccinic acid (C3H6O4S), folic acid (C19H19N7O6), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), 3-(4,5-dimethylazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), and N-hydroxy succinimide were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Cell culture reagents, fetal bovine serum (FBS), Dulbecco's Modified Eagle Medium (DMEM, high glucose), penicillin-streptomycin, trypsin/EDTA, Dulbecco's phosphate buffer saline (PBS), Atto 680 fluorescence dye, and 4,6-diamidine-2-phenylindole dihydrochoride (DAPI, blue) cell staining kits were purchased from Gibco BRL (Carlsbad, CA, USA). NIH 3T3 cells (animal fibroblast cells) and KB cells (oral squamous cancer cells) were purchased from the Korean Cell Line Bank. All other chemicals used in this study were analytical grade and used without further purification.
Synthesis of mono-dispersed gold nanoparticles
Gold nanoparticles were prepared according to the method reported by Anshup et al. . Briefly, 100 ml of 0.05% HAuCl4 was added to a 250-ml round-bottom flask and boiled. Under rapid stirring, 3.5 ml of sodium citrate (1%) was added and further rapidly stirred for 15 min. After stirring for 30 min, the solution was gradually cooled to room temperature. After 15 min, the liquid was extracted and filtrated by a 0.22-mm filter paper. The prepared gold nanoparticles were dissolved and purified by centrifugation and double re-precipitation from distilled water.
Preparation of hydrophilic gold nanoparticles
Mercaptosuccinic acid (0.16 M) was dissolved in 70 ml of water into a 250-ml three-neck round-bottom flask containing gold nanoparticles. The clear solution was stirred for 3 h under nitrogen. When the reaction was complete, water was removed by vacuum, and the residue was mixed with 4 ml of water and transferred to a centrifuge tube. Subsequently, 20 ml of water was added to the mixed solution, and the precipitated product was separated by centrifugation (3,000 rpm for 15 min) and washed with water. The prepared mercaptosuccinic acid-conjugated gold nanoparticles were dissolved and purified by centrifugation and double re-precipitation from distilled water.
Immobilization of folic acid on the GM surface
Preparation of GF conjugated to Atto dyes
GF conjugated to Atto fluorescent dye (GFA) were obtained by reacting GF (1.5 × 10−3 mol/l), dissolved in a carbonate buffer (pH 8), with an Atto dye (4.8 × 10−5 mol/l) dissolved in water at room temperature. The concentration of Atto was measured by an UV-Vis spectrophotometer (680 nm) to confirm the number of fluorophore molecules conjugated to each ligand molecule .
Characterization of surface-modified gold nanoparticles
In order to confirm the presence of folic acid and Atto dye on the surface of gold nanoparticles, UV-Vis absorption spectra were recorded from aqueous dispersions at room temperature using a Hitachi U-3000 spectrophotometer (Hitachi, Tokyo, Japan). Transmission electron microscopy (TEM, Philips, Amsterdam, Netherlands; CM 200 TEM, applied operation voltage: 120 kV) was used to observe the morphology of nanoparticles. To obtain the samples for the TEM observation, the gold nanoparticles were diluted with distilled water and deposited on Formvar-coated 400 mesh copper grids (Agar Scientific, Essex, UK). After drying the nanoparticle-fluid thin film on the copper grid, a thin carbon film, approximately 10 to 30 nm in thickness, was deposited on the nanoparticles fluid film. The hydrodynamic diameter and size distribution of the gold nanoparticles were determined by dynamic light scattering (DLS) using a standard laboratory-built light scattering spectrometer with a BI90 particle sizer (Brookhaven Instruments Corp., Holtsville, NY, USA). The system was equipped with a vertically polarized incident light of 514.5 nm supplied by an argon ion laser (LEXEL Laser, model 95, Cambridge Lasers Laboratories, Inc., Fremont, CA, USA).
KB and NIH-3T3 were used as target and control cells, respectively. The cells were cultured routinely at 37°C in a humidified atmosphere containing 5% CO2 in a polystyrene dish containing 10 ml of MEM or DMEM medium, supplemented with 10% fetal bovine serum and 1% penicillin streptomycin G sodium (PGS). The medium was changed every third day. For subculture, the cells were washed twice with PBS and incubated with a trypsin-EDTA solution (0.25% trypsin, 1 mM EDTA) for 10 min at 37°C to detach the cells. Complete media were then added to the polystyrene dish at room temperature to inhibit the effects of trypsin. The cells were washed twice by centrifugation and resuspended in complete media for reseeding and growing in new culture flasks. To observe the morphology, the cells were seeded at a concentration of 1 × 105/ml in a 10-ml petri dish and incubated for 3 days with a media containing GM or GF at a concentration of 0.2 mg/ml. The morphology of the adhered cells was observed by an optical microscope (Nikon Eclipse TS100, Tokyo, Japan).
To assess the cytotoxic effects of GF, after 2 days of culture, the KB and NIH-3T3 cells were suspended in PBS with a cell density of 1 × 105 cells/ml. Subsequently, 200 μl of a cell suspension was mixed with a 100-μl assay solution [10 μl calcein-AM solution (1 mM in DMSO) and 5 μl propidium iodide (1.5 mM in H2O) mixed with 5 ml PBS] and incubated for 15 min at 37°C. The cells were then examined by fluorescence microscopy (Carl Zeiss, Axioplan 2, Jena, Germany) with 490 nm excitation for the simultaneous monitoring of viable and dead cells.
Intracellular uptake of gold nanoparticles
To examine the cellular uptake of nanoparticles via fluorescence and confocal laser microscopy, the cells were seeded at a concentration of 1 × 105/ml in a 10-ml petri dish and incubated for 1 day. The medium was then replaced with a medium containing GM and GF at a concentration of 50 μg/ml and incubated for the time (1 to 6 h) required for internalization of the nanoparticles into the cells. The cells were then washed three times with Dulbecco's PBS (D-PBS), and images were obtained using fluorescence and confocal laser microscopes. The fluorescence images were obtained using an Olympus IX70 fluorescence microscope (Olympus Corp., Tokyo, Japan) equipped with a cooled charge-coupled device (CCD) camera. The images were processed using DVC view software (version 2.2.8, DVC Company). A Zeiss LSM 410 confocal laser-scanning microscope (brightness 700 cd/mm2, Zeiss, Oberkochen, Germany) was used to obtain the confocal images. The position and integrity of the internalized GF nanoparticles were evaluated by confocal microscopy using 4,6-diamidine-2-phenylindole dihydrochoride (DAPI, blue) as a marker. The cell nuclei were stained by the addition of DAPI solution (10 μL) with suitable mixing and incubated for 10 min. In order to track the GF nanoparticles, F-conjugated GM and DAPI (488 nm) were added to the cells. The stained cells were washed at least three times with 1 ml of fresh DMEM medium, and images were then obtained by confocal laser microscopy . To quantify the intracellular uptake of the nanoparticles, cells were grown in a 24-well culture dish with approximately 105 cells in 1 ml of medium. After incubation at 37°C for 20 h, the cells were reseeded with a culture medium containing GF at a concentration of 2 × 104 cell/ml. The gold nanoparticles uptaken by the cells were quantified by inductively coupled plasma (ICP). The cells were washed with PBS, detached, resuspended, counted, centrifuged down, and the cell pellets were dissolved in 37% HCl aqueous solution at 79°C to 80°C for 30 min. The samples were diluted to a final gold concentration of 1.0 to 4.0 μg/ml. The experiment was repeated three times and the results were averaged.
The cell proliferation experiment was performed in triplicate, and the results were expressed as mean ± standard deviation (SD). The Student's t test was employed to assess the statistical significance of differences in the results. A difference was considered statistically significant at p < 0.05.
Results and discussion
Atomic percentage of GE and GF calculated from the ESCA survey scan spectra
Evaluation of cytotoxicity
Evaluation of intracellular uptake
Mercaptosuccinic acid-coated gold nanoparticles were successfully conjugated with folic acid. The formation of folic acid-immobilized GM was confirmed by UV and XPS. The size of GF, as determined by DLS, was about 25 nm. The GF nanoparticles exhibited no cytotoxicity on the control (NIH-3T3) and target cells (KB). In vitro cell experiments showed that folic acid-immobilized GM can specifically recognize oral squamous cancer cells and emit intense fluorescence images, as well as exhibiting more efficient intracellular uptake into KB cells compared to animal skin cells (NIH3T3). These results suggest that GF has a potential for optical imaging applications and for the treatment of squamous cancer cells.
This study was supported by the Basic Research Laboratory Program (No. 2011-0020264) and General Research Program (2013 R1A1A 2005148) from the Ministry of Education, Science and Technology of Korea.
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