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yi Zheng
Fuzhou University
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Article
Journal of
Biomedical Nanotechnology
State Key Laboratory of Photocatalysis on Energy and Environment, Research Institute of Photocatalysis,
Fuzhou University, Fuzhou, 350002, P. R. China
2
Facult de Medicine, Groupe en Sciences des Radiations, Universit de Sherbrooke, Sherbrooke, Qubec, J1H 5N4, Canada
Gold nanoparticles (GNPs) sensitize biomolecules to radiation in two ways: by locally increasing the radiation energy
absorbed and by modifying the sensitivity of the target biomolecules to radiation. Taking DNA as the biological target,
we present the first investigation of the latter chemical mechanism of radiosensitization by irradiating thin films made of
GNP-DNA complexes with 10 eV electrons. Naked GNPs of 5 and 15 nm diameters were synthesized and electrostatically
bound to DNA. Damage to the GNP-DNA complexes were analyzed, as a function of electron fluence, by electrophoresis.
In identical 5-monolayer films, the yield of DNA damage, as well as the enhancement factor due to the presence of 5 nm
positively-charged nanoparticles, increased with rising ratio of GNPs to DNA up to 1:1. In comparison, increasing the ratio
of negatively-charged 15 nm GNPs to DNA did not increase damage. As verified by XPS and zeta potential measurements, the binding of plasmid DNA to the surface of the two sizes of GNPs varies owing to the characteristics of the GNP
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surface and electrostatic interaction.
The results
indicate that
strong binding
of GNPs
to DNA could significantly influence
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On:
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2014
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the efficiency of the chemical radiosensitization mechanism. This mechanism appears to be an important component of
Copyright: American Scientific Publishers
the overall process of GNP radiosensitization and should be considered when modeling this phenomenon. Our results
suggest that small size GNPs (diam. 5 nm) are more efficient radiosensitizers compared to larger GNPs when delivered
into cancerous cells, where their action should be cell-cycle dependent.
INTRODUCTION
Within the continuous development of cancer therapies,
radiotherapy remains a fundamental treatment for primary tumors and metastases.1 An efficient modality to
improve radiotherapy consists of introducing a radiosensitizing agent into cancer cells, which increases the local
dose deposited in the tumor relative to the dose deposited
in healthy tissues.2 Recently, gold nanoparticles (GNPs)
have attracted considerable interest as a potential novel
radiosensitizer capable of increasing the radiation dose
within cancer cells.3
Investigations on GNP radiosensitization have been
undertaken in vivo and in vitro, as well as with molecular
models.3, 4 In most studies, the increase in radiosensitivty is
measured as an enhancement factor (EF), expressed as the
478
doi:10.1166/jbn.2015.1922
Yao et al.
479
Yao et al.
EXPERIMENTAL DETAILS
were also conducted with films of GNP-II-DNA complexes having a thickness of 10 ML, to determine any
GNPs of two diameters were synthesized. GNPs-I were
significant differences in the results obtained with the two
prepared by reduction of HAuCl4 by NaBH4 .20 GNPsfilm thicknesses. Since GNPs are excellent conductors, the
II were prepared according to the method of Frens.24
increase in film thickness to 10 ML did not cause any
Freshly prepared GNPs were characterized by TEM and
measurable additional film charging in the LEE irradiation
zeta potential analysis. The GNP solutions were deposited
experiments.
on 230 mesh Formvar-carbon(FC)-coated copper grids and
In the present study, we applied the same LEE irradiimaged with a Tecnai G2 F20 S-TWIN, FEI TEM. The
ation procedure as previously described.27 After lyophylTEM was operated in the high resolution mode with an
ization, the films were transferred directly to the UHV
accelerating voltage of 200 KV and a beam current of
chamber of the LEE irradiator. The DNA films were then
4200 A. The TEM images were recorded with a digibombarded with an incident electron beam of 6 nA and
tal camera (AMT Version 2.25). The size distribution of
electron energy of 10 0.5 eV, during exposures of 5 to
GNPs was analyzed by ImageJ (Version 1.41) counting at
60 s. The time-response of the yields was expressed in
least 200 particles in the TEM image. The average diameterm of the fluence (i.e., the total number of electrons hitter was 5 3 nm and 15 2 nm for GNPs-I and GNPs-II,
ting the target). To obtain a single fluence-response curve,
respectively. Assuming a cubic close packing of the gold
36 samples had to be irradiated with 6 identical experiatoms (0.288 nm diameter), the concentration of GNP-I
ments for a given fluence. A fluence-response curve was
and GNP-II solutions were calculated to be 3.9 104
recorded for each preparation with different GNP numbers
and 8.69 106 nmol/l, respectively. The zeta potenand sizes for total of 48 independent experiments. Cortials () of the GNPs was measured by dynamic light scatrespondingly, 6 control samples were used to record the
tering analysis (Zeta sizer 3000HSA), at room temperature
zero fluence data point in the exposure curve. After irradia25 C, with an Ag electrode. was automatically calcution, samples were removed from the vacuum chamber and
lated from electrophoretic mobility based on the Smoluwere immediately dissolved in 20 ul ddH2 O. The recovery
chowski equation, v = E/, where v is the measured
of DNA was approximately 90%.
electrophoretic velocity, is the viscosity, is the elecThe different forms of DNA were separated by 1% neutrical permittivity of the electrolytic solution and E is the
tral agarose gel electrophoresis ran in TAE buffer (40 mM
electric field. The recorded mean value of for GNP-I and
by Publishing Technology
to: Leon
Tris acetate,
1 mMSanche
EDTA, pH 8.0) at 100 V for 7 min
II was + 10.25 and 33.97 mV,Delivered
respectively.
IP: 132.210.107.48 On: Wed, 26 Nov 2014 17:12:23
and
75
V
for
90
min.
pGEM-3Zf(-) plasmid DNA (3197 base
pairs,
Promega)
Copyright: American Scientific Publishers Both the gel and the DNA samples
were prestained with SYBR Green I (Molecular Probes),
was extracted from E. coli DH5 and purified with QIAfil10,000 for gel and 100 for samples, respectively. After
ter Plasmid Giga Kit (Qiagen). The DNA was eluted in TE
electrophoresis, gels were scanned with the STORM860
(10 mM Tris and 1 mM EDTA) buffer and diluted in dissystem (Molecular Dynamics) using the blue fluorescence
tilled deionized water (dd H2 O) for subsequent manipulamode at an excitation wavelength of 450 nm. The percenttion. Agarose gel electrophoresis analysis showed that 95%
age of each form was obtained from Image Quant analysis.
of the purified plasmid DNA was in the supercoiled form
The weaker binding of SYBR Green I to the supercoiled
and the remaining 5% was in the concatemeric (1%) and
form of DNA compared to nicked circular (SSB) and linnicked circular form (4%). The absolute amount of plasear (DSB) configurations was corrected by a normalization
mid DNA was determined by measuring the UV absorpfactor of 1.4.
tion at 260 nm. The ratio of UV absorption at 260 nm to
The binding interaction of GNPs with DNA was verified
280 nm was 1.96, indicating fairly low impurity content
by XPS. The XPS measurements were conducted using a
in the DNA.25 The amount of protein was determined by
commercial system (Thermo Scientific ESCALAB 250).
absorption at 280 nm; corresponding to less than 10% by
The apparatus was operated at an emission current of 6 mA
weight of protein remaining with the DNA.26 GNP soluproducing a monochromatized Al K beam in a chamber
tions were mixed with 248 ng/l DNA to form GNP-DNA
at a base pressure of 38 1010 mbar. The neutralizing
complexes with different molar ratios (R).
electron gun was turned on in the low energy mode with
Films of DNA and GNP-DNA complexes composed of
an emission current of 100 mA to eliminate the charging
GNPs-I and GNPs-II were deposited on a tantalum subof the samples during X-ray irradiation. The hemispheristrate (99.9% Goodfellow) by lyophilisation. All samples
cal electron energy analyzer input axis was normal to the
had a thickness of 5 monolayers (ML). Since the diamesample surface. The high-resolution Au 4f spectra were
ter of 5 nm of GNPs-I is much smaller than the 15 nm
recorded with a passing energy of 20 eV and energy steps
thickness of a 5 ML film, these GNPs were completely
embedded in the DNA. Experiments with GNPs-I were
of 0.1 eV. No signal of Au 4f was detected on the surface of GNP-I-DNA. The Au 4f spectrum of GNP-I-DNA
therefore conducted only with the 5 ML films. However,
from geometrical considerations, it can be readily seen that
(1:1) film could only be obtained after Argon ion etching
GNPs-II, with their larger diameter, are not necessarily
of the film surface. The Argon gun was operated at 2 KV
completely embedded in the 5 ML film. Thus, experiments
and 1 A for 60 s. Argon etching removed approximately
480
Yao et al.
Ratio of
GNP:DNA
DSB(%)
GNP % (mass)
SSB
DSB
Supercoiled
46 5
2.9 0.2
46 11
54
107
161
268
536
75 6
133 4
181 20
216 14
201 15
4.8 0.1
8.6 0.2
11.1 0.8
12.8 0.9
11.5 0.5
80 8
134 6
182 18
222 31
207 3
182
363
69 8
62 4
3.5 0.5
3.6 0.1
75 10
63 3
0
GNPs-I
1:5
2:5
3:5
1:1
2:1
GNPs-II
1:5
2:5
0.4
0.3
0.2
0.1
16
SSB(%)
Yield
12
5.0
8
SSB
DSB
4.5
supercoiled
GNP-II:DNA
4.0
slope
87
84
3.5
3.0
1.4
EF
Supercoiled(%)
Enhancement factor
GNP-I:DNA
90
2.5
2.0
1.2
1.5
81
0
10
15
20
0.2
0.4
ratio of GNP-II to DNA
1.0
0.0
0.4
0.8
1.2
1.6
2.0
481
Yao et al.
this is not the case for complexes constructed with largerin the lyophilized aliquot. Contact alone of GNPs-I with
size GNPs. Even if GNPs-II of 15 nm have 27 times more
DNA increases SSB almost linearly with a rising ratio
gold atoms than GNPs-I, both sizes produce the same EF
of GNP number to DNA, up to 22.2% at 2:1. A similar
for SSB at a ratio of R = 02, while for DSB the EF with
observation has recently been made in the construction of
GNPs-II is even smaller than that of GNPs-I. As the ratio
double stranded DNA-GNP biosensors, where a direct coris increased to 0.4, the EF surprisingly decreases for senrelation was found between DNA denaturation and surface
sitization with GNPs-II. This observation is not obvious to
coverage on GNPs.28 The non-irradiated GNPs-II films
exhibit a different behavior. The presence of GNPs-II in
interpret, since from purely geometrical considerations, the
the irradiated film results in a much lower formation of
surface area of GNPs-II is 9 times larger than that of GNPsSSB, showing a peak of 8.2% at an R of 1:15 and a
I. Thus, a larger portion of the DNA should be in contact
decrease to 6.4% at an R of 2:5. Beyond R = 04 (not
with the GNPs-II and, at an identical ratio, a larger EF (i.e.,
shown) the yields remain fairly constant, up to R = 2.
a higher radiosensitivity) would be expected from GNPs-II.
These results suggest that the two types of GNPs may
That is unless the surface area of GNPs related to its size
interact differently with DNA.
is not a relevant factor governing the nanoscale mechanism
At identical exposure with 10 eV electrons, the two
of chemical sensitivity to LEE irradiation.
types of GNPs continue to exhibit totally different sensiTo further elucidate the different sensitivities to DNA
tivity trends. For GNPs-I, irradiation with 10 eV electrons
damage on the two sizes of GNPs, the formation of SSB
produces SSB and loss of supercoiled DNA, with percentand loss of supercoiled DNA was recorded as a function
ages increasing linearly with the number of added GNPs
of GNP ratios with and without exposure of 56 1011
electrons in UHV; the results are shown in Figures 3(A)
(i.e., R), while the increase in yield rate (the slope of the
(GNP-I) and (B) (GNP-II). These yields, at corresponding
dashed linear fit in Fig. 3(A)) is about 37% and 54% larger
ratios, are also plotted for samples from the non-irradiated
than those of the non-irradiated sample. Thus, the pressolution, shown in Figure 3. The curves drawn from the trience of GNPs-I near DNA leads to an enhancement of
angles indicate that adding GNP to DNA in solution does
radiosensitivity.
not appreciably perturb the molecule. However, when an
On the other hand, irradiated films of GNPs-II contain a
aliquot of the DNA-GNP complex solution is lyophilized
maximum yield of 11.8% SSB at 1:15, but DNA radiosenon a Ta substrate and kept under UHV for 24 h, the helix
sitivity is only marginally increased; i.e., by approxiby Publishing
to: Leon(Fig.
Sanche
unwinds leading to SSB withoutDelivered
any irradiation
(i.e., full Technology
mately 23.6%
3(B)) compared to non-irradiated
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circles in Fig. 3). Under identical conditions
as
those
of
films.
When
R
is
increased
to 2:5, the yield drops to
Copyright: American Scientific Publishers
the irradiated samples, the damage to the non-irradiated
close to the level observed at an R of 1:40. The addition
samples increases as a function of added number of GNPs
of more GNPs of larger size (15 nm) evidently does
12
24
SSB (%)
SSB (%)
32
16
8
non-irradiated
solution
91
92
irradiated
84
77
solution
70
non-irradiated
irradiated
63
84
linear fit
0.0
0.5
88
1.0
1.5
2.0
0.00
0.05
0.10
0.15
0.20
0.4
Figure 3. Comparison of SSB formation and loss of supercoiled DNA for GNP-DNA complexes under different conditions: films
deposited on tantalum bombarded in UHV by 56 1011 electrons of 10 eV (), non-irradiated in UHV () and kept in solution
(). The percentage of different DNA forms is shown as a function of the ratio of the number of GNPs to DNA. Panels (A) and
(B) correspond to the results obtained with 5-ML films of GNPs-I-DNA and GNPs-II-DNA containing identical amounts of DNA
(320 ng).
482
Yao et al.
Counts (a.u.)
483
Yao et al.
inner Au atoms in the nanospheres, as recorded in a preGNPs and this sensitivity would result in a high level
vious study.21 The other component labelled A in Figure 4
of radiosensitization by LEEs, which constitute a major
is attributed to the surface Au atoms, which bind elecsecondary product of high energy radiation. Since during
trostatically with DNA, and have higher binding energies
transcription the DNA-RNA duplex adopts the A form,
of 85.3 and 88.8 eV, respectively. By integrating the area
we can further speculate that small (5 nm diam.) GNP
under the deconvoluted peaks of the GNPs-I-DNA specshould be more genotoxic and chemically radiosensitiztrum, we found that 43% of the signal arises from the
ing during the S cycle of the cell (i.e., during DNA
shifted BE spectra, whereas 57% of the signal comes from
replication).
the non-perturbed line. Within the experiment measurement errors, the shifted spectra value is in good agreement
CONCLUSION
with the corresponding percentage of surface gold atoms
The DNA damage of GNP-DNA complexes induced by
for GNPs-I, i.e., 45%. This correlation suggests that the
10 eV electrons was investigated to study the chemical
surface of GNPs-I are entirely surrounded by DNA, i.e.,
mechanism of DNA radiosensitization induced by GNPs.
the contact area of GNPs-I interacting with DNA is nearly
These data showed that positively-charged 5 nm GNPs-I,
100%. Furthermore, the splitting of the Au 4f lines into a
which bind strongly to DNA, resulted in a nearly consimple doublet in the GNPs-I DNA spectra suggests unistant increase of damage as a function of nanoparticleform binding of the nanospheres to DNA, possibly at the
DNA ratio. The optimum EF for radiosensitization reaches
PO4 site.
4.5 when the amounts of 5 nm diameter GNPs are added
In contrast, characteristics of doublet peaks are not
to DNA in a ratio of 1:1. Negatively-charged GNPs of
found in the GNPs-II-DNA spectrum; instead, more than
15 nm diameter appear to bind weakly and randomly
two doublet peaks are required to obtain a good fit.
to DNA, resulting in much less radiosensitization comAt least 2 additional pairs of peaks are needed, as shown
pared to the smaller GNPs. Our results suggest that the
in Figure 4. The broad peak of the Au 4f line indicates a
nature of the GNP binding to DNA is an important parammore complex interaction of DNA on the surface of GNPseter in the chemical mechanism of radiosensitivity and
II. These have a negative , can hardly bind to DNA and
resultant damage induced. Since this chemical sensitivhence could scatter over different sites causing smearing
ity of DNA to GNPs occurs in the A-form of DNA, if
of the Au 4f peak, owing to different binding nature of the
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the GNPsto:reach
cell nucleus they could be cell-cycle
sites. Thus, the existence of complex
binding
of GNPs-II Technology
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26
Nov
2014
17:12:23
dependent. In order
to possess cell-cycle specificity, the
with DNA could be explained by the non-uniform
site
disCopyright: American Scientific Publishers
GNP released in the cytoplasm must be sufficiently small
tribution of GNPs-II in DNA film compared to that of
(diam. 5 nm). Thus, in in vivo and in vitro studies of
GNPs-I.
GNP radiosensitization for cancer treatment, there appears
According to our results, the chemical mechanism of
to be two reasons to transport GNPs of small size into
radiosensitization would have quite a different behaviour
cancerous cells (e.g., by liposomal encapsulation36): to
compared to physical mechanisms. For example, the study
increase the action of the chemical mechanism of radiosenof GNP radiosensitization induced by X-rays radiation in
sitivity and penetration of GNPs into the nucleus to
solution, which relies on both chemical and physical mechreach DNA.
anisms, show that the largest GNP lead to the highest EF
Recently, more elaborate models, such as the Local
for DNA damage.34 Monte Carlo simulations also indiEffect Model of McMahon et al.17 have linked the relcate that GNPs with larger diameters contribute to a larger
ative biological effectiveness (RBE) to physical paramedose enhancement and generation of secondary electrons.35
ters of X-ray absorption by GNPs, taking into account the
These results are contradictory to our results, which indisignificant contribution of short-range electrons to dose
cate (Fig. 2) that increasing size does not increase the EF;
i.e., size dependence may not be the same for the chemical
inhomogeneity in the system. The chemical mechanism
mechanism of radiosensitization, which rely essentially on
of radiosensitivity revealed in the present study should
nanoscale binding of the GNP to DNA, where different
improve the explanation of the processes which increase
surface charges affect the interaction with DNA in the film.
RBE, particularly those arising from the action of shortThe zeta potential of GNPs-II prepared by citrate reduction
range LEEs.
indicates a relatively high negative surface charge comAcknowledgment: Financial support for this work was
pared to a positive surface charge of GNPs-I prepared with
provided by the Canadian Institutes of Health Research
sodium borohydride. Since DNA is a negatively charged
(MOP81356), the China Award Program of Minjiang
biomolecule, it is expected that electrostatic binding of
Scholar Professorship, National Basic Research Program
DNA on the surface of GNPs-II to be fairly weak and
of China (973 Program: 2007CB613306), the Program
random compared to that of the slightly positive GNPs-I,
for Changjiang Scholars and Innovative Research Team
involving different binding levels as indicated in the XPS
in University (PCSIRT0818), and the NNSF of China
spectrum (Fig. 4). Furthermore, DNA in the A form would
(20973039).
be much more sensitive to strong electrostatic binding of
484
Yao et al.
REFERENCES
485