Increased apoptotic potential and
Blackwell Publishing Asia
dose-enhancing effect of gold nanoparticles
in combination with single-dose clinical electron
beams on tumor-bearing mice
Meng-Ya Chang,1 Ai-Li Shiau,2 Yu-Hung Chen,1 Chih-Jui Chang,1,5 Helen H-W Chen,3,4,6 and Chao-Liang Wu1,6
1
Department of Biochemistry and Molecular Biology, National Cheng Kung University Medical College, 1 Dashiue Road, Tainan 701, Taiwan, 2Department of
Microbiology and Immunology, National Cheng Kung University Hospital 138 Sheng-Li Road, Tainan 701, Taiwan, 3Institute of Clinical Medicine, National Cheng
Kung University Medical College, Tainan, 701, Taiwan, 4Department of Radiation Oncology, National Cheng Kung University Hospital, Tainan, 701, Taiwan
(Received December 7, 2007/Revised February 27, 2008/Accepted March 10, 2008/Online publication April 11, 2008)
High atomic number material, such as gold, may be used in con-
junction with radiation to provide dose enhancement in tumors. In
the current study, we investigated the dose-enhancing effect and
apoptotic potential of gold nanoparticles in combination with single-
dose clinical electron beams on B16F10 melanoma tumor-bearing
mice. We revealed that the accumulation of gold nanoparticles was
detected inside B16F10 culture cells after 18 h of incubation, and
moreover, the gold nanoparticles were shown to be colocalized
with endoplasmic reticulum and Golgi apparatus in cells. Furthermore,
gold nanoparticles radiosensitized melanoma cells in the colony
formation assay (P + 0.02). Using a B16F10 tumor-bearing mouse
model, we further demonstrated that gold nanoparticles in
conjunction with ionizing radiation significantly retarded tumor
growth and prolonged survival compared to the radiation alone
controls (P < 0.05). Importantly, an increase of apoptotic signals was
detected inside tumors in the combined treatment group (P < 0.05).
Knowing that radiation-induced apoptosis has been considered a
determinant of tumor responses to radiation therapy, and the
length of tumor regrowth delay correlated with the extent of
apoptosis after single-dose radiotherapy, these results may suggest
the clinical potential of gold nanoparticles in improving the outcome
of melanoma radiotherapy. (Cancer Sci 2008; 99: 1479–1484)
R adiation dose enhancement by high atomic number (Z)
materials has long been investigated. In theory, loading
high Z materials into the tumor could result in greater
photoelectric absorption within the tumor than in surrounding
tissues, and thereby enhance the dose delivered to a tumor
during radiation therapy. At least 20 years ago, it was noted in
vitro that this effect might be employed to enhance radiotherapy
for cancer.(1) Accumulating studies have demonstrated the
dose enhancement caused by high Z materials in kilovoltage
beams(2–4) and in megavoltage beams.(5–8) Moreover, enhanced
cell killing was also observed when cells were irradiated
adjacent to high Z materials by kilovoltage X-rays.(9–12) In
clinical practice, electron beams from linear accelerators
have increasingly taken the place of kilovoltage X-ray beams for
skin and subcutaneous tumors because they offer distinct
advantages in terms of dose uniformity in the target volume and in
minimizing the dosage to deeper tissues.(13) Although kilovoltage
beams could maximize tumor dose enhancement, it has
technical restrictions. The use of kilovoltage X-rays produces
significant dose heterogeneity inside the target tumor.(4,14)
To be clinically useful, a radiosensitizer and/or dose enhancer
should significantly increase the therapeutic ratio and should
be readily available, easily utilized, and non-toxic. Gold (Au;
Z = 79) or nanogold (gold nanoparticles, AuNP) showed dose-
doi: 10.1111/j.1349-7006.2008.00827.x
© 2008 Japanese Cancer Association
enhancing effects in cell experiments,(15) the murine model,(16)
and through Monte Carlo calculations.(17) Gold nanoparticles
have been actively investigated in a wide variety of biomedical
applications due to their biocompatibility and ease of conju-
gation to biomolecules.(18–21) Besides, nanoparticles have the
advantages of small size (1–100 nm) and ability to evade the
immune system,(22,23) and also have been shown to preferentially
accumulate in tumors.(24–28)
While previous studies have primarily examined the dose
enhancement factor by Au, it is also known that radiation-
induced apoptosis is a significant component of radiation-induced
cell death. Consequently, modulating the apoptotic response and
thereby the radiosensitivity is of interest.(29–34) Therefore, in the
current study, we investigated the dose-enhancing effect and
apoptotic potential of gold nanoparticles in combination with
single-dose clinical electron beams on B16F10 melanoma
tumor-bearing mice.
Materials and Methods
Preparation of AuNP. AuNP were prepared as previously
described with slight modifications.(35) All glassware used in
these preparations was thoroughly cleaned in aqua regia (3 parts
HCl and 1 part HNO3), and all solutions were made using
18-MΩ-deionized, 0.22-μm-filtered water. Briefly, 50 mL of
HAuCl4 (1 mM) was reduced with sodium citrate (38.8 mM,
5 mL) by boiling with vigorous stirring for 10 min. The resulting
burgundy suspension was cooled, sterile-filtered, and stored
in glass bottles at room temperature or 4°C. The AuNP were
spherical and well-dispersed with an approximate diameter of
13 nm confirmed by a transmission electron microscopy. The
particle concentration was approximately 10.72 nM (180 μg/mL)
quantified by a maximum absorption at 520 nm. The prepared
nanoparticle solution could be concentrated by centrifugation
and was stable for at least 6 months.
Cells and mice. Murine B16F10 melanoma cells were cultured
in Dulbecco’s modified Eagle’s medium supplemented with
50 μg/mL gentamicin, 2 mM L-glutamine, and 10% cosmic calf
serum (Hyclone, Logan, UT, USA) at 37°C in an atmosphere of
5% CO2. Female C57BL/6 mice (6–8-week-old) were obtained
from the Laboratory Animal Center of the National Cheng Kung
University. Animals were maintained in specific pathogen-free
5
Present address: Institute of Molecular and Cell Biology, Tzu Chi University,
No 701, Zhongyang Road, Section 3, Hualien, 97004, Taiwan.
6
To whom correspondence should be addressed.
E-mail: wumolbio@mail.ncku.edu.tw; helen@mail.ncku.edu.tw
Cancer Sci | July 2008 | vol. 99 | no. 7 | 1479–1484

Fig. 1. Visualization of gold nanoparticles (AuNP) inside B16F10 cells.
AuNP localization in cells was visualized by silver enhancement or by
fluorescence labeling of AuNP. (a) The localization of AuNP was
determined by silver enhancement, and (b) the localization of AuNP-.
Alexa Fluor 594 conjugates were detected directly under a microscope
(bar = 50 μm; original magnification 200×). BF, Bright field, DAPI, 4′-6-
diamidino-2-phenylindole.
animal care facilities under isothermal conditions with regular
photoperiods. All animal experiments were performed following
the guidelines approved by the Laboratory Animal Care and Use
Committee of the National Cheng Kung University.
Visualization of AuNP. AuNP localization was visualized by
silver enhancement or by fluorescence labeling of AuNP. B16F10
melanoma cells were seeded on cover glass at 37°C for 6 h, and
the medium was replaced by culture medium with or without
10 nM AuNP or AuNP-Alexa Fluor 594 conjugates (Alexa
Fluor 594 was purchased from Invitrogen, Eugene, OR, USA).
Eighteen hours post-AuNP addition, cells were washed with
phosphate-buffered saline (PBS), the localization of AuNP was
determined by silver enhancement of AuNP according to the
manufacturer’s instructions (Sigma, St Louis, MO, USA), and
the localization of AuNP-Alexa Fluor 594 conjugates was
detected directly under a microscopy with an Olympus DP1T
digital camera system (Olympus Optical, Tokyo, Japan).
Live-cell endoplasmic reticulum (ER) and Golgi labeling. ER-Tracker
Red dye and BODIPY TR Ceramide Golgi Tracker (Molecular
Probes, Carlsbad, CA, USA) were used, respectively, for live-cell
ER and Golgi labeling according to the manufacturer’s instructions.
Animal studies.
Tumors. C57BL/6 mice were inoculated subcutaneously (s.c.)
in the thigh with syngeneic mouse melanoma B16F10 cells
(1 × 106) suspended in 0.1 mL of PBS at day 0, and at day 7,
1480
visible nodules developed at all injection sites with approximate
tumor volumes of 50–90 mm3. Groups of four to seven tumor-
bearing mice were injected intravenously (i.v.) with 200 μL of
200 nM AuNP in phosphate buffer (PB), or with 200 μL of PBS
via the lateral tail vein. All mice were monitored for tumor
growth and survival as previously described.(36)
Irradiation. Approximately 24 h post-AuNP injection, a 1-inch
diameter tumor region of the leg was irradiated with 6 MeV
electrons using a Varian 2100C linear accelerator (Varian Medical
Systems, Palo Alto, CA, USA) under normoxic conditions. The
delivered dose was 25 Gy per mice. Animals were anesthetized
with pentobarbital intraperitoneally (90 μg/g body weight).
Biodistribution of AuNP. Groups of three tumor-bearing mice
were injected with AuNP or with PBS via the lateral tail vein.
Twenty-four hours postinjection, blood and tissues were excised.
Samples were pooled and dried at 105°C until the weight
remained constant, and then were homogenized into powder. For
AuNP detection, 0.5 g of tissue samples was used. Six mililiters
of concentrated HCO3 was added into each sample, and
incubated at room temperature until the samples were completely
nitrated. After nitration, 2 mL of H2O2 was added. Once the
solution became clear, samples were then filtrated to remove cell
debris. The resulting samples were then analyzed for Au
concentration using atomic absorption detection (National Sun
Yat-Sen University, Kaohsiung, Taiwan).
Clonogenic survival. The effectiveness of the combination of
AuNP and ionizing radiation was assessed by clonogenic assays.
The B16F10 melanoma cell lines were treated with AuNP
(10 nM) for 18 h and then exposed to different doses of ionizing
radiation. Briefly, cells were irradiated with using a linear
accelerator (Varian 2100C; Varian, Palo Alto, CA, USA) at room
temperature in T-25 flasks. After treatment, cells were trypsinized
and counted. Known numbers were then replated and returned
to the incubator to allow macroscopic colony development.
Colonies were counted after 7 days, and the plating efficiency
and surviving fraction for given treatments were calculated
based on the survival of non-irradiated cells treated with the
vehicle or AuNP alone.
Detection of apoptosis. Apoptotic activity was analyzed on the
basis of terminal deoxynucleotidyl transferase-mediated
deoxyuridine triphosphate nick end labeling (TUNEL) assay.
Tumors from groups of three tumor-bearing mice were excised
6 h postradiotherapy,(37) and embedded in OCT compound (Sakura
Finetek USA, Torrance, CA, USA). Five-micrometer thick cryostat
sections from each representative specimen were obtained and
fixed, and then processed with the DeadEnd Fluorometric
TUNEL System (Promega, Madison, WI, USA) according to the
manufacturer’s protocol. The positive TUNEL signals were
counted under a microscopy with an Olympus DP1T digital
camera system (Olympus Optical). DAPI was used as the nuclear
counter stain. Apoptotic cells were calculated by averaging the
number of positive TUNEL signals from eight fields with
highest density of TUNEL signals in each section.
Statistical analysis. Data were expressed as mean ± standard
error of the mean (SEM). The survival analysis was done using
the Kaplan–Meier survival curve and the log-rank test. Other
statistical differences were assessed with Student’s t-test.
Statistical significance was set at P < 0.05.
Results
Visualization of AuNP in B16F10 cells. To detect the AuNP
inside cultured cells, the localization of AuNP was visualized by
silver enhancement (Fig. 1a) or by direct observation under a
fluorescent microscopy (Fig. 1b). Our data demonstrated that
after 18 h of incubation, AuNP could be detected inside the
B16F10 cells. Moreover, we also found that the AuNP did not
colocalize with the cell nucleus (Fig. 1b).
doi: 10.1111/j.1349-7006.2008.00827.x
© 2008 Japanese Cancer Association

Fig. 2. Gold nanoparticles (AuNP) colocalized with endoplasmic reticulum
(ER) and Golgi in cells. B16F10 cells were cultured with AuNP for 20 h.
The localization of AuNP was determined by silver enhancement, and
the (a) ER-Tracker Red dye and (b) BODIPY TR Ceramide Golgi Tracker
were used for live-cell ER and Golgi labeling (bar = 50 μm; oiginal
magnification 200×; BF, bright field).
Fig. 3. Biodistribution of gold nanoparticles (AuNP) in mice. (a)
Twenty-four hours after AuNP injection, tissues of tumor-bearing mice
were excised, processed, and used for AuNP detection using atomic
absorption detection. (b) Silver staining of AuNP inside a tumor. Twenty-
four hours after AuNP injection, tumors were excised and paraffin-
embedded. Five-micrometer thick sections from each representative
specimen were obtained and then processed with the silver enhancement
kit (bar = 200 μm). PBS, phosphate-buffered saline.

AuNP revealed colocalization with ER and Golgi apparatus in

B16F10. As the AuNP revealed a non-uniform distribution in the
cytoplasm of B16F10 cells, to investigate the possibility that the
subcellular localization of AuNP is in ER or Golgi, live-cell ER or
Golgi staining was used in addition to the silver enhancement of
AuNP. Our results indicated that AuNP were localized in ER
(Fig. 2a) and Golgi apparatuses (Fig. 2b) in B16F10 20 h after
incubation.
Biodistribution of Au 24 h postintravenous injection of AuNP in
tumor-bearing mice. To detect the biodistribution of AuNP 24 h
postinjection, blood and tissues were excised and analyzed for
Au concentration using the atomic absorption detection
(Fig. 3a). The result showed that at 24 h following injection,
a notable accumulation of AuNP inside tumor tissues was
detected. The tumor-to-tumor surrounding muscle gold ratio
was 6.4:1. Nevertheless, higher concentrations of AuNP were
also found in spleen and liver, which indicated that AuNP were
also uptaken by the reticuloendothelial system.
In agreement with this biodistribution data, sliver enhancement
of AuNP in the tumor biopsies also revealed the presence of
AuNP inside tumor tissues (Fig. 3b).
AuNP radiosensitized melanoma cells. B16F10 cells were Fig. 4. Gold nanoparticles (AuNP) radiosensitized melanoma cells.
B16F10 cells were treated with AuNP (10 nM for 18 h) and assessed for
treated with AuNP and assessed for radiosensitization by radiosensitization by clonogenic cell survival immediately after irradiation.
clonogenic cell survival immediately after irradiation (Fig. 4). For the survival curves, each data point represents the average of three
Our result revealed that AuNP radiosensitized B16F10 melanoma independent experiments each plated in triplicate ± SD (solid line,
cells in the colony formation assay. control; dotted line, 10 nM AuNP; *P = 0.02).

Chang et al. Cancer Sci | July 2008 | vol. 99 | no. 7 | 1481

© 2008 Japanese Cancer Association
Fig. 5. Antitumor effects of the combination treatment of gold
nanoparticles (AuNP) and radiotherapy in tumor-bearing mice. C57BL/6
mice were inoculated subcutaneously with B16F10 cells (1 × 106) at day
0. At day 7, tumor-bearing mice were injected intravenously with 200 μL
of 200 nM AuNP, or with 200 μL of phosphate-buffered saline (PBS) 24 h
before irradiation (25 Gy/mouse). Mice were monitored for (a) tumor
growth and (b) survival (n = 4–7; *P < 0.05). RT, radiotherapy.

Antitumor effects of the combination of AuNP with radiotherapy

in tumor-bearing mice. To investigate whether the combination of
AuNP and radiotherapy resulted in better antitumor effects in
terms of tumor growth and survival than radiation alone, a
syngeneic melanoma model was used in the animal study. Our
result revealed that the tumor growth was both retarded in mice
receiving either radiation alone or receiving AuNP followed
by radiation (Fig. 5a) compared to the controls with no
radiation. More importantly, tumor volume in the combination
therapy group was significantly smaller compared with that in
radiation alone group (P < 0.05), whereas administration of
AuNP or PBS alone did not exert any antitumor effect on tumor-
bearing mice (Fig. 5a).
Furthermore, the Kaplan–Meier survival curves of the treated
groups are illustrated in Figure 5b. The survival of mice with radia-
tion and AuNP combination therapy was significantly longer
than that of the radiation alone mice (P < 0.05; log-rank test).
Apoptosis in tumors with AuNP and radiotherapy combination
therapy. Increasing evidence has indicated the important
contributing role of apoptosis in radiation-induced cell death
and for apoptosis as a determinant of radiosensitivity.(30–32) In the
present study, we examined whether apoptosis was associated
with the antitumor effects of combination therapy. As shown in
Figure 6a, the extent of apoptosis observed in TUNEL-stained
Fig. 6. Apoptotic activity was analyzed by terminal deoxynucleotidyl
transferase-mediated deoxyuridine triphosphate nick end labeling
(TUNEL) assay. Tumors were excised from mice 6 h postradiotherapy.
Five-micrometer thick representative cryostat sections were obtained
and then processed with the TUNEL System. (a) Positive TUNEL straining
was observed under a fluorescent microscopy. (b) Quantitative analysis.
Apoptotic cells were calculated by averaging the number of positive
TUNEL signals from eight fields with highest density of TUNEL signals in
each section (n = 8; *P < 0.05; original magnification 40×). RT, radiotherapy.
cryosections was found higher after a single-dose radiotherapy
compared to that in the no radiation controls. Noticeably, the
number of apoptotic cells detected was significantly higher in
the AuNP and radiation combination group than that in the
radiation alone group (P < 0.05). The quantitative results are
represented in Figure 6b.

1482 doi: 10.1111/j.1349-7006.2008.00827.x

© 2008 Japanese Cancer Association
Discussion
In the present study, our important finding was that in
combination of AuNP with clinical electron beams in radiotherapy,
an increase of apoptotic potential was observed in TUNEL-
stained cryosections of the tumors in mice. Knowing that
radiation-induced apoptosis has been considered a determinant
of tumor responses to radiation therapy, and the length of
tumor regrowth delay correlated with the extent of apoptosis
after single-dose radiotherapy,(30) this result indicates the
clinical potential of AuNP in improving the outcome of cancer
radiotherapy.
Using sliver enhancement and fluorescent staining, we were
able to determine the localization of AuNP inside B16F10 cells.
We detected the presence of AuNP in cells after 18 h and 42 h
of incubation, and furthermore, we found that AuNP were
colocalized with ER and Golgi staining rather than with the
nucleus of cells. It has been shown that continuous ER stress
results in apoptotic cell death;(38) therefore, the accumulation of
AuNP in ER and Golgi may also contribute to the increase of
the apoptotic potential of cells postirradiation.
Recently, considerable investigations have been made in
exploring the role of apoptosis in cellular radiation responses.
Although the contribution of cellular apoptotic potential to overall
radiosensitivity and tumor responses to radiotherapy has been
debated for several years, increasing evidence suggests that
restoring the tumor apoptotic potential may have considerable
therapeutic impact.
In the current study, we demonstrated the dose-enhancing
effect of AuNP in conjunction with single-dose clinical electron
beams in a B16F10 melanoma tumor-bearing model, in which
the tumor growth was retarded and the mice survival was
prolonged. These effects were consistent with the results for a
previous mammary tumor model;(16) however, many less AuNP
were injected intravenously into the mice in this study (1 g/kg
compared to 2.7 g/kg). Besides, in the current study, the irradiation
time-point, 24 h post-AuNP injection, was used instead of a
time-point of 2 min postinjection. Accumulation of unlabeled
nanoparticles within tumor occurred through the enhanced
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