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Abstract
The treatment of ovarian cancer has traditionally been intractable, and required novel
approaches to improve therapeutic efficiency. This paper reports that thio-glucose bound gold
nanoparticles (Glu-GNPs) can be used as a sensitizer to enhance ovarian cancer radiotherapy.
The human ovarian cancer cells, SK-OV-3, were treated by gold nanoparticles (GNPs) alone,
irradiation alone, or GNPs in addition to irradiation. Cell uptake was assayed using inductively
coupled plasma atomic emission spectroscopy (ICP-AES), while cytotoxicity induced by
radiotherapy was measured using both 3-(4,5)-dimethylthiahiazo
(-z-y1)-3,5-di-phenytetrazoliumromide and clonogenic assays. The presence of reactive oxygen
species (ROS) was determined using CM-H2-DCFDA confocal microscopy and cell apoptosis
was determined by an Annexin V-FITC/propidium iodide (PI) kit with flow cytometry. The
cells treated by Glu-GNPs resulted in an approximate 31% increase in nanoparticle uptake
compared to naked GNPs ( p < 0.005). Compared to the irradiation alone treatment, the
intracellular uptake of Glu-GNPs resulted in increased inhibition of cell proliferation by
30.48% for 90 kVp and 26.88% for 6 MV irradiation. The interaction of x-ray radiation with
GNPs induced elevated levels of ROS production, which is one of the mechanisms by which
GNPs can enhance radiotherapy on ovarian cancer.
S Online supplementary data available from stacks.iop.org/Nano/22/285101/mmedia
(Some figures in this article are in colour only in the electronic version)
1. Introduction In 2009, 21 550 new cases were diagnosed and 14 600 women
died of ovarian cancer in the United States alone. Only
Ovarian cancer is particularly insidious in nature and is the
leading cause of death among gynecological malignancies [1]. 20% of patients are diagnosed early enough for treatment
to be effective [2]. Traditional methods of chemotherapy,
8 These authors equally contributed to this article.
9 Address for correspondence: Department of Obstetrics and Gynecology, surgery, and radiotherapy can control cancer symptoms;
Qilu Hospital of Shandong University, 107 Wenhuaxi Road, Jinan, 250012, however, these procedures lack targeting specificity [3]. In
Shandong, People’s Republic of China particular, radiotherapy covers all cancer cells within its
0957-4484/11/285101+08$33.00 1 © 2011 IOP Publishing Ltd Printed in the UK & the USA
Nanotechnology 22 (2011) 285101 F Geng et al
radiation field, inevitably subjecting abdominal organs, such Fisher scientific, USA) was used for surface characterization
as the liver, kidneys, and small bowel, to lethal radiation. of Glu-GNPs to determine the elemental composition of each
For radiotherapy to be effective, a therapeutic mechanism is Glu-GNP.
needed in order to enhance the cancer killing effects while
minimizing cytotoxicity to surrounding tissues. Current data 2.3. Cell lines and culture conditions
provide insight on gold nanoparticle (GNP) enhanced radiation SK-OV-3 (HTB-77), an epithelial ovarian cancer cell line, was
therapy on ovarian cancer cells. purchased from American Type Culture Collection (ATCC)
Previous studies using metallic nanoparticles show and cultured in RPMI Medium 1640 (GIBCO, Invitrogen
promising tumor-killing potential over conventional methods Corporation) supplemented with 10% heat-inactivated fetal
of chemotherapy, radiation, or surgery [4]. Gold is an ideal bovine serum (FBS, GIBCO). Cells were cultured in water
material that ensures biocompatibility while being capable of jacketed CO2 incubators (Thermo Fisher Scientific Forma®
forming reactive oxygen species when irradiated [5–11]. Using Series II, USA) at 37 ◦ C with 95% (v/v) air and 5% (v/v) CO2
1.9 nm GNPs, Hainfeld et al [12] obtained dose enhancement in a humidified atmosphere.
ratios of at least two when tumor bearing mice were irradiated
with 250 kVp x-rays. Our previous research has shown that 2.4. Cell uptake of GNPs and Glu-GNPs
Glu-GNPs significantly increased cell uptake by various types
SK-OV-3 cell were cultured in 6 cm dishes. When the cells
of cancer tissues compared to bare nanoparticles [12, 13].
reached 70% confluence, GNPs and Glu-GNPs were added
Glu-GNPs showed a 1.5–2.0 fold enhancement in growth
into the medium respectively for a final concentration of
inhibition compared to GNPs alone in prostate and breast
5 nM. Because FBS might have an impact on binding and
cancer treatment [12]. Its effect on ovarian cancer remains
internalization of GNPs, we used FBS-free medium when
to be investigated. In this paper, we investigate Glu-GNPs
GNPs were incubated with the cells. In brief, we removed
enhanced target cytotoxicity of radiation on ovarian cancer the culture medium containing FBS and washed the cells with
cells. PBS buffer twice. The cells were then cultured with FBS-
free medium and treated with GNPs. After the treatment,
2. Materials and methods the medium containing FBS was used to replace FBS-free
2.1. Chemicals medium. After incubation at different intervals (1, 2, 4, 8,
12, 24, 48, and 96 h), the cells were collected and then re-
Gold (III) chloride trihydrate (HAuCl4 ·3H2 O, G4022-1g), suspended into PBS for a final volume of 5 ml. The number of
sodium borohydride (NaBH4 , 452882), sodium citrate cells was counted using a hemocytometer. 5 ml of 20% HNO3
iribasic dehydrate (C6 H5 Na3 O7 ·2H2 O, S4641-500G), 1-thio- was added into each sample to lyse the cells. The gold mass
D-glucose (GLU, T6375-1G) and polyethylene glycol (PEG in the lysis solution was measured by ICP-AES. The number
5000, 11124) were purchased from Sigma-Aldrich, USA. All of GNPs was calculated via the gold mass, and the number
the materials were dissolved in deionized water purified by the of GNPs in the lysis solution divided by the number of cells
Milli- Q Biocel system (ZMQS50F01, Millipore, USA). provided a quantitative measurement of GNP cell uptake.
Three sub-steps were involved in GNP synthesis. (i) 3.2 ml SK-OV-3 ovarian cancer cells were seeded at approximately
of 25 mM HAuCl4 solution was added to 60 ml of deionized 2 × 103 per well of a 96-well tissue culture plate and incubated
water in an ice bath under moderate stirring. (ii) 4 ml of 26 mM overnight. The medium was replaced by fresh medium
NaBH4 was then added as a reducing agent to obtain naked containing different concentrations of Glu-GNPs (0, 1, and
GNPs. (iii) 22.4 ml of naked GNP solution was added to two 5 nM). 24 h later, the medium containing GNPs was removed,
tubes: the first containing 4 ml of 20 mM 1-thio-β -glucose the cells were washed twice with PBS, and new medium with
FBS was added. The cells were then divided into two groups,
and the second containing 4 ml of 38.8 mM sodium citrate
one without irradiation, and the other was followed by either
solution. Thio-glucose formed a covalent bond with the GNPs
(i) irradiation with low-energy 90 kVp x-rays (Faxitron x-
while sodium citrate was electrostatically bound to the GNPs
rays); or (ii) irradiation with high-energy 6 MV photons, by
to form functionalized Glu-GNPs and neutral naked GNPs,
a medical linear accelerator (Varian 23EX linear accelerator,
respectively. Both the naked GNPs and the Glu-GNPs were
USA), each with a total dose of 10 Gy. After irradiation, cells
dialyzed for two days to remove any free sodium citrate or thio-
were incubated at different intervals (24, 48, 72, and 96 h,
glucose before these solutions were used for the experiments.
respectively). The cells without nanoparticles or irradiation
The gold (Au) concentration was tested by inductively served as controls. For all experiments, cell viability was
coupled plasma atomic emission spectroscopy (ICP-AES) measured using the 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-
(IRIS INTREPID II XSP). The morphology of gold phenytetrazoliumromide (MTT) (Amresco, 0793-1G) assay.
nanoparticles was characterized using transmission electron The results for cellular survival in response to Glu-GNPs
microscopy (TEM) (JEM-100CX, Japan). The size distribution with and without radiation were determined using the Opsys
of GNPs was determined by dynamic light scattering MR™ 96-well microplate reader (CB372, DYNEX, USA) and
(DLS) (LB-550, HORIBA Jobin Yvon, USA). X-ray expressed as the absorbance at 490 nm at the indicated points
photoelectron spectroscopy (XPS) (ESCALAB 250, Thermo in time.
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Nanotechnology 22 (2011) 285101 F Geng et al
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Nanotechnology 22 (2011) 285101 F Geng et al
Figure 1. Characterizing Glu-GNPs. (A) TEM picture of GNPs alone; (B) and (C) the schematic of GNPs coated with thio-glucose; (D) the
size distribution of Glu-GNPs measured by DLS; (E) characterizing Glu-GNPs using XPS.
toward 5 Gy 6 MV irradiation, showing survival rates ranging and 20 Gy, the inhibitory rates were 4.23%, 12.6%, 37.5%,
from 79.6% for x-ray alone to 61.3%, 60.0%, and 58.2% for 83.75%, and 100%, respectively.
the combined treatments, respectively. A significant decrease
in survival rate was observed, averaging 10.6% ( P < 0.01)
compared to controls (figure 2(D)). 3.5. Intracellular reactive oxygen species (ROS) concentration
Cell colony formation assay was also used to determine
the sensitivity of Glu-GNPs enhanced radiation. 6 MV To investigate the effect of x-ray induced ROS on cancer cells,
irradiation with doses of 2.5, 5, 10, 15, and 20 Gy induced we used CM-H2-DCFDA, a fluorescence-based probe that
inhibitory rates of 9.9%, 26.1%, 39.6%, 56.8%, and 76.2%, detects the intracellular production of ROS. CM-H2-DCFDA
respectively. If 5 nM Glu-GNPs were added before irradiation, passively diffuses into cells, and becomes deacetylated by
the same doses of irradiation produced inhibitory rates of intracellular esterases. It is subsequently oxidized to a
11.7%, 30.6%, 61.4%, 92.8%, and 100%, respectively ( P < fluorescent product in the presence of intracellular ROS where
0.007) (figure 2(F)). Similar enhancement ratios were observed the fluorescence indicates the level of intracellular oxidative
for 90 kVp irradiation groups. Cells subject to 90 kVp
stress. Figure 3 showed that Glu-GNPs enhance the production
radiation doses of 2.5, 5, 10, 15, and 20 Gy experienced growth
of intracellular ROS when irradiated with 8 Gy x-rays. 90 kVp
inhibition rates of 17.7%, 35.6%, 54.5%, 78.6%, and 95.5%,
irradiations at 8 Gy induced an approximately 5.1-fold increase
respectively. When combined with 5 nM Glu-GNPs, the same
dose of radiation-induced cellular inhibitory rates were found in basal CM-H2-DCFDA fluorescence (figure 3(C)), which
with 26.2%, 55.2%, 91.7%, 100%, and 100%, respectively was enhanced to an 8.3-fold increase by adding 5 nM Glu-
( P < 0.007) (figure 2(E)). A comparison of enhancement GNPs before x-irradiation ( p < 0.05) (figure 3(D)). Similarly,
rates induced by either 6 MV or 90 kVp irradiations is shown 6 MV irradiation at 8 Gy induced a 3.4-fold increase for
in figure 2(G). Higher sensitization ratios were achieved for irradiation alone and a 7.8-fold increase for irradiation plus
90 kVp irradiation than 6 MV irradiation. When 5 nM Glu- GNPs in basal CM-H2-DCFDA fluorescence ( p < 0.05)
GNPs were subject to irradiation doses of 2.5, 5, 10, 15, (figures 3(E) and (F)).
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Nanotechnology 22 (2011) 285101 F Geng et al
Figure 2. Study of the interactions between nanoparticles and SK-OV-3 cells. After culturing with either GNPs or Glu-GNPs for 24 h,
SK-OV-3 cancer cells were used for analyzing the uptake and cytotoxicity effects of nanoparticles, or for treating by the irradiation (5 Gy):
(A) cell uptakes of GNPs versus Glu-GNPs; (B) cytotoxicity induced by GNPs and Glu-GNPs measured by the MTT assay; (C) survival rates
for groups with 90 kVp irradiation (5 Gy) plus Glu-GNPs measured by the MTT assay; (D) survival rates for groups with 6 MV irradiation
(5 Gy) plus Glu-GNPs measured by the MTT assay. GNPs enhanced radiotherapy measured by the colony formation: the inhibition rate
induced by Glu-GNPs plus different doses of 90 kVp irradiation (E) or 6 MV irradiation (F); (G) comparison of the enhancement of using
GNPs with either 90 kVp irradiation or 6 MV irradiation.
3.6. Apoptosis detection by flow cytometry induced a significant increase in apoptosis (18.57 ± 1.44%)
compared to irradiation alone (14.35 ± 0.90%, P = 0.003)
To assess the effect of GNPs on 6 MV x-ray induced apoptosis, (figure 4). These data indicate that one mechanism of the radio
dual staining of cells with Annexin V-FITC and PI was used enhancement effect of GNPs is due to increased cell apoptosis.
to quantitatively distinguish apoptotic cells from normal and
necrotic cells. Dots in the lower-right (LR) quadrant represent
3.7. Glu-GNPs alter cell cycle distribution
early stage apoptotic cells and dots in the upper-right (UR)
quadrant represent late stage apoptotic cells. Therefore, the Treatment of SK-OV-3 cells with 5 nM Glu-GNP for 2 h
sum of LR and UR represents the apoptotic rate. Before induced an increase of cells in the G2/M phase and a decrease
irradiation, cells in the Glu-GNP group experienced similar of cells in the G0/G1 phase when compared with the control
levels of apoptosis to the control group (9.26 ± 2.16% versus cells (figures 4(F)–(H)). GNPs arrested cells at G2/M, the
7.06 ± 2.49%, P = 0.13). However, exposure to 6 MV radiosensitive phases of the cell cycle, and thereby enhanced
irradiation caused significant increases in the apoptosis of SK- the radiation sensitivity of SK-OV-3 cells. In this study, 9.28%
OV-3 cells compared to controls (14.35 ± 0.90% versus 7.06 ± of the untreated control cells were in the G2/M phase, and
2.49%, P = 0.017). Glu-GNPs subject to 6 MV irradiation Glu-GNP increased the fraction of cells in the G2/M phase
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Nanotechnology 22 (2011) 285101 F Geng et al
Figure 3. The cellular fluorescence changes resulting from intracellular ROS production were measured by a confocal microscope. After
culturing with or without Glu-GNPs for 24 h, SK-OV-3 cancer cells were treated with irradiation:. (A) control; (B) 5 nM Glu-GNPs;
(C) 90 kVp irradiation (8 Gy); (D) 90 kVp irradiation (8 Gy) + 5 nM Glu-GNPs; (E) 6 MV irradiation (8 Gy), and (F) 6 MV irradiation
(8 Gy) + 5 nM Glu-GNPs.
to 20.52%. For the G0/G1 phase, 43.35% for the control experiments used glucose as a targeting ligand to coat the
was decreased to 27.82% for the cells treated with Glu-GNPs surface of GNPs. Since cancerous cells metabolize much
(5 nM) for 2 h. faster, they uptake glucose at significantly higher rates,
allowing for selective internalization of Glu-GNPs [12]. The
faster cancer cells grow, the faster the metabolism rate, and
4. Discussion thus the more uptake of glucose. It is difficult to accurately
make the comparison between cells’ uptake of Glu-GNPs
Radiation enhancement by metallic nanoparticles has been
by ovarian cancer cells and by normal ovarian cells using
widely reported both in vivo [7] and in vitro [8–11]. In
in vitro tests because in vitro tests cannot properly present
animal testing, GNPs significantly increased the survival rate the growth rate of the normal ovarian cells. However, our
of mice bearing subcutaneous EMT-6 mammary carcinomas in vivo data indicated that the biodistribution of Glu-GNPs
after receiving 250 kVp x-rays [7]. Meanwhile, Rahman et al in cancer tissue is ten times higher than those in normal
reported the radiation enhancing effects of kilovoltage x-rays ovarian and uterus tissue (unpublished data) (see supporting
and megavoltage electrons in bovine endothelial cells [9]. Our data available at stacks.iop.org/Nano/22/285101/mmedia). In
previous study showed that the radiation efficiency of 200 kVp this study, uptake concentrations reached peak levels between
x-rays significantly increased for breast cancer and prostate 24 and 48 h, then diminished thereafter. Glucose significantly
cancer cells containing internalized Glu-GNPs [12, 13]. Our increased the localized uptake of GNPs by SK-OV-3 cells and,
experiments are the first worldwide to demonstrate that Glu- moreover, allowed nanoparticles to stay internalized longer
GNPs enhance the sensitivity of ovarian cancer cells to 6 MV in the cytoplasm. Based on observed cell uptake kinetics, a
photons and 90 kVp x-rays. radiotherapy regimen was formulated to administer irradiation
A major engineering challenge is the delivery of 24 h after GNPs were injected into the bloodstream.
nanoparticles to the targeted tumor site. Various approaches In developing GNP enhanced radiotherapy, most studies
for targeted delivery have been investigated [15, 16]. Our have focused on low-energy radiation because high atomic
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Nanotechnology 22 (2011) 285101 F Geng et al
Figure 4. The cell apoptosis induced by Glu-GNPs induced radiotherapy was measured by the flow cytometry dot plots of Annexin
V-FITC/PI dual staining. After culturing with or without Glu-GNPs for 24 h, SK-OV-3 cancer cells were detected by flow cytometry:
(A) control, (B) Glu-GNPs alone, (C) x-ray alone, (D) Glu-GNPs + x-ray, (E) blank. The flow cytometric analysis of the cell cycle induced
by Glu-GNPs: (F) control without treatment; (G) treatment with Glu-GNPs; and (H) comparison of the changes of cell cycle between the
control group and the cells treated with Glu-GNPs for 2 h.
number ( Z ) materials such as gold preferentially absorb present study, to our knowledge, we are the first group to
kilovoltage x-rays compared to higher-energy megavoltage demonstrate that Glu-GNPs at very low concentration (5 nM)
radiation [17]. Our present study demonstrates that Glu- with 6 MV x-ray irradiation can produce a high level of
GNPs achieved superior enhancement ratios at 90 kVp than intracellular ROS to kill ovarian cancer cells (figure 3).
6 MV. However, orthovoltage x-rays are limited in therapeutic These ROS lead to higher elevated levels of oxidative stress
applications, only effective for cancer near the body’s surface. manifesting as increased levels of apoptosis compared to
Megavoltage x-rays are far more common in radiotherapy, irradiation alone (figure 4). The results indicate that increased
particularly for deep-seated tumors such as ovarian cancer. ROS formation when radiation interacts with GNPs is a key
Hence, for radio-therapeutic treatment of ovarian cancer it mechanism that mediates cancer cell apoptosis.
seems far more practical to use GNPs to enhance megavoltage Another intriguing aspect of GNPs’ behavior is their
radiotherapy. Data in figures 2(D) and 4(F) show that Glu- disproportionate cytotoxicity toward cancer cells. In our
GNPs enhance radiation sensitivity toward 6 MV photons by previous study, MCF-7 cancer cells and MCF-10A normal
24% in SK-OV-3 cells. The effect of amplified MV x-rays cells (non-cancerous) were made to internalize the same
on cell apoptosis cannot be singularly attributed to high- Z concentration of GNPs with the expectation that they would
materials alone, additional mechanisms must be considered, induce similar irradiation cytotoxicities [12]. However, after
such as GNP interactions with the cellular cycle [18]. being exposed to identical radiation doses, the viability of
Ionizing radiation is known to generate ·OH radicals the cancer cells decreased significantly (about 40%) while no
through radiolysis of water molecules. These free radicals significant changes were observed in the normal cells [13].
react rapidly with multitudes of biological macromolecules, These results provide convincing evidence that GNPs are
such as nucleic acids, proteins, and lipids to induce nucleic involved in cellular mechanisms apart from ROS enhancement.
base damage, DNA–protein cross-links, lipid peroxidation, and Turner et al reported that metallic materials may arrest cells
protein degradation [17, 20]. These factors are a potential at the G2/M phase, the most radiosensitive phase of the cell
trigger for radiation-induced apoptosis [21, 22]. Although cycle [23], and thus disproportionately increase the sensitivity
GNPs alone at a very high concentration (10 μM) were of cancer cells toward radiation. Zhang et al reported that Glu-
reported to generate a significant level of ROS [19], this GNPs trigger activation of the CDK kinases, leading cancer
concentration is too high for clinical applications. In the cells to accumulate in the G2/M phase. Consequently, after
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Nanotechnology 22 (2011) 285101 F Geng et al
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This project was supported by the grants to Beihua Kong expressing PLCgamma1 using surface-enhanced Raman
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National Natural Science Foundation of China. The project [16] Huang X et al 2006 Cancer cell imaging and photothermal
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