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Chemical Radiosensitivity of DNA Induced by Gold

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Article
Journal of
Biomedical Nanotechnology

Copyright 2015 American Scientific Publishers

All rights reserved
Printed in the United States of America

Vol. 11, 478485, 2015

www.aspbs.com/jbn

Chemical Radiosensitivity of DNA Induced by

Gold Nanoparticles
Xiaobin Yao1 , Chuanna Huang1 , Xiaoping Chen1 , Yi Zheng1
, and Leon Sanche2
1

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
Delivered
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Technology
to: Leon
Sanche
surface and electrostatic interaction.
The results
indicate that
strong binding
of GNPs
to DNA could significantly influence
IP:
132.210.107.48
On:
Wed,
26
Nov
2014
17:12:23
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.

KEYWORDS: Gold Nanoparticles, Chemical Radiosensitivity, DNA.

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

Author to whom correspondence should be addressed.

Email: Yizheng@fzu.edu.cn
Received: 5 July 2013
Accepted: 4 January 2014

478

J. Biomed. Nanotechnol. 2015, Vol. 11, No. 3

magnitude of the measured variable (e.g., cell death, tumor

growth, survival or molecular damage) with GNPs divided
by the magnitude obtained without GNPs, under identical experimental conditions. In in vivo studies of tumorbearing mice, an increased survival rate was observed
when the animals were injected with 36 mg kg1 of
GNPs 24 h prior to radiotherapy,5 whereas no synergistic
effect appeared for GNP coated with dithiolated diethylenetriaminepentaacetic gadolinium (Au@DTDTPA:Gd) and
radiation.6 At the cellular level, radiosensitisation by GNPs
has been studied in various tumor. Most EFs for an in
vitro dose of cell mortality were in the range of 13.7,7, 8
depending on the type of radiation, energy of radiation
and the size of the GNPs. An exceptionally high EF of
20 was found in the BAEC cell line with 1.9 nm GNP
and 80 kV X-ray.8 Together with in vivo studies in tumorbearing mice,912 the biological studies indicated that the
presence of GNPs caused the generation of reactive oxygen species and oxidative stress, while displaying different sensitivities in cell cycle kinetics including an increase
1550-7033/2015/11/478/008

doi:10.1166/jbn.2015.1922

Yao et al.

Gold Nanoparticles and Chemical Radiosensitivity

of sub G1 cell population or accumulation in G2/M cell

mechanism from the other. One way to probe for damcycle.4
age induced only by the chemical mechanism may be to
The physical rationale for GNP radiosensitization is
irradiate a GNP-DNA complex, with a source of radiaessentially based on the greater mass energy absorption
tion, which does not produce secondary electron emission
coefficient of gold; i.e., the presence of heavy-metal GNPs
by interacting with the GNPs. Electrons with energies
in cancer cells can increase the absorption of ionizing
around 10 eV or lower do not produce a significant
radiation energy and potentially increase damage and cell
amount of secondary electrons when interacting with a
death. However, considering this aspect alone it is difsolid metal target.18 On the other hand, such secondary
electrons constitute an important component of the secficult to explain the existing data. In the kilovolt range,
ondary species created by ionizing radiation in biological
gold absorbs between 10 and 150 times more energy per
media.19
unit mass from X-rays compared to soft tissue.13 How5, 6, 912
7, 8
and in vitro are
Zheng et al. reported that electrostatic binding of GNPs
ever, the EFs of GNPs in vivo
much smaller than expected based only on the increase of
enhanced the yields of single-strand breaks (SSBs) and
dose deposition in gold. The discrepancy between physidouble-strand breaks (DSBs) by a factor of 2.5 after expocally predicted increases in X-ray energy deposition and
sure to high-energy electron irradiation.20 The contribution
of secondary low-energy electrons (LEEs) emitted by the
experimental observations in cell killing and survival rates
GNPs to the damage was subsequently revealed by binding
indicates that the radiosensitivity of GNPs cannot be excluligands of different lengths to the nanospheres.21 Ligands
sively due to the difference in energy absorption by gold
of varying length bound to the GNPs attenuated the LEEs
and tumor tissue; other parameters must be taken into
produced on the surface of GNPs and thus reduced the
consideration.
numbers of LEEs reaching DNA and therefore reduced the
Among the theoretical calculations of dose absorbed
amount of additional damage. These results showed good
by GNPs and the effect on cell killing,1416 McMahon
et al. developed a Monte Carlo model which takes
correlation with LEE attenuation lengths except when the
into account the structure of energy deposition in the
DNA was in direct contact with the GNPs. When the EFs,
nanoscale vicinity of GNPs, rather than a macroscopic
induced by naked GNPs electrostatically bound to plasmid
view of dose. The simulation showed good agreement with
DNA, were measured after irradiation by 1, 10, 100 and
experimentally observed cell killing by X-ray irradiation
60,000 eV electrons, the largest EF value (3.2) was found
by Publishing
22 Leon Sanche
that a detailed
under- Technology
At this energy, essentially no secondary elecand GNPs.17 Thus, it appearsDelivered
for 10 eV.to:
IP: 132.210.107.48 On: Wed, 26 Nov 2014 17:12:23
standing of GNP radiosensitivity may
require
developtrons
are
expected
to be emitted from the GNPs, but there
Copyright: American Scientific Publishers
ment of molecular models at the nanoscopic level, which
is a maximum in DNA damage owing to the formation of
would ideally contain all of the physical, chemical and
transient negative ions (TNIs).23 Therefore it was speculated that TNI parameters could be modified by the presbiological parameters involved in the radiosensitization
ence of the metal so as to increase bond breaking within
process.
DNA in the vicinity of the GNPs. In other words, the large
Since cell survival without mutation generally depends
enhancement of damage in DNA-GNPs complexes bomon genome integrity, DNA is often used as a simple molecbarded by 10 eV electrons could only be caused by the
ular model to investigate directly induced radiation damchemical mechanism of radiosensitization.
age, while excluding the biological response of the cell,
In the present work, we investigated the chemical mechsuch as DNA repair. Within the perspective of such a
anism of radiosensitization by modifying different parammodel, a radiosensitizer usually increases DNA damage
eters which could influence radiosensitization of DNA
by modifying the morphology and/or the chemical strucby GNPs. Thin films of pure plasmid DNA and plasmid
ture of the molecule (i.e., in modified DNA, the secondary
DNA-GNPs complexes were bombarded by 10 eV elecspecies produced by the primary radiation induces more
trons under ultra-high vacuum (UHV) conditions. With an
damage). Thus, GNPs are somewhat unusual radiosensitizidentical amount of DNA in each film, we measured the
ers in the sense that they are expected to increase damage
formation of SSB, DSB and the loss of the supercoiled
not only via modification of the DNA, but also by increasconfiguration as a function of electron fluence. In these
ing the absorption of ionizing radiation energy close to
experiments, different binding interaction of GNPs with
the DNA. This enhancement of energy absorption in turn
DNA was achieved by changing the size of the GNPs in
leads to a considerable increase in the production of short
GNP-DNA complexes. The surface of gold in contact with
range secondary electrons that have a high probability of
the plasmid was varied by changing the number of GNPs,
damaging the DNA molecule. In the text, we refer to the
of the same size, bound to the DNA. The damage was meafirst and second mechanisms as chemical and physical
sured by electrophoresis and GNP surface properties were
mechanisms of radiosensitization, respectively.
characterized by transmission electron microscopy (TEM),
Although the chemical mechanism is common to many
zeta potential and XPS measurements. The results indiradiosensitizers, it does not appear to have been indecate that chemical sensitivity depends on the electrostatic
pendently investigated in the case of GNPs. This lack of
binding ability of GNPs to DNA.
information may be due to the difficulty in separating one
J. Biomed. Nanotechnol. 11, 478485, 2015

479

Gold Nanoparticles and Chemical Radiosensitivity

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

J. Biomed. Nanotechnol. 11, 478485, 2015

Yao et al.

Gold Nanoparticles and Chemical Radiosensitivity

2.4 nm (0.4 /s 60 s = 24 nm) of surface material and

exposed GNPs to vacuum. It indicated that GNPs-I are
covered by DNA in the original film. For all measurements, the energy scale of the XPS spectra was calibrated
on the standard C 1s binding energy line of 284.6 eV.

Table I. Yields per 1015 electron molecule, for the formation

of SSB, DSB and loss of supercoiled DNA induced by 10 eV
electrons in different DNA films deposited on a tantalum substrate. The error is the standard deviation obtained from three
identical experiments. The second column is the mass percentage of GNPs in a 5 ML film containing 320 ng of DNA.

RESULTS AND DISCUSSION

Ratio of
GNP:DNA

Freshly-prepared films of GNP-DNA complexes with the

same amount of DNA per ML were irradiated by 10 eV
electrons for different periods of time. The percentages of
SSB, DSB and supercoiled DNA in the irradiated GNPsI-DNA and GNPs-II-DNA samples with R of 1:5 are displayed numbers of incident electrons in Figure 1. These
exposure-response curves behave similarly to those previously published for 10 eV electron impact on pure DNA
films of the same thickness.23 At an identical R of 1:5,
the GNPs-I-DNA suffered relatively more damage compared to GNP-II-DNA. The percentage yield of formation

DSB(%)

GNP % (mass)

SSB

DSB

Supercoiled

46 5

2.9 0.2

46 11

5
4
10
7
16
1
26
8
53
6

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

18
2
36
3

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

of SSB, DSB and loss of supercoiled DNA per electron

are obtained from the initial slope of the corresponding
exposure curves. The effective yields at various ratios are
summarized in Table I.
The EF, defined as the yield of damage from a
GNP-DNA complex normalized to that of DNA without
GNPs, illustrates the sensitivity of DNA to the presence
of GNPs. The EFs for formation of SSB and DSB as a
function of
ratio
of GNPs to DNA are displayed in
Delivered by Publishing Technology
to: the
Leon
Sanche
IP: 132.210.107.48 On: Wed,
26 Nov
2014the
17:12:23
Figure
2. With
same amount of DNA in the film, the
Copyright: American Scientific
Publishers
EF reached
a maximum at a ratio of 1:1 for GNP-I and was
saturated thereafter. Since the amount of DNA was kept the
same (i.e., the number of targets being sensitized by GNPs
was constant), on average one GNP per DNA appears sufficient to achieve maximum radiosensitization with the 5 nmdiameter nanospheres. As seen from the insert of Figure 2,

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

Number of incident electrons (1011)

0.2
0.4
ratio of GNP-II to DNA

1.0
0.0

0.4

0.8

1.2

1.6

2.0

ratio of GNP-I to DNA

Figure 1. Exposure-response curve for GNP-DNA complexes
irradiated with 10 eV electrons and molar ratio of GNPs to DNA
(R) of 1:5 for GNP-I () and GNP-II (
). Each data point corresponds to the mean value of the percentage yields of DSB,
SSB and loss of the supercoil configuration of 6 samples
standard deviation.
J. Biomed. Nanotechnol. 11, 478485, 2015

Figure 2. Enhancement factor (EF) for the formation of SSB,

DSB and loss of supercoiled DNA induced by 10 eV electron
impact on GNP-I-DNA complexes as a function of molar ratio
of GNP to DNA. The insert shows the EF of GNP-II-DNA complexes (open symbols).

481

Gold Nanoparticles and Chemical Radiosensitivity

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
IP: 132.210.107.48 On: Wed, 26 Nov 2014 17:12:23
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

Super coiled (%)

Super coiled (%)

irradiated

84
77
solution
70

non-irradiated
irradiated

63

84

linear fit
0.0

0.5

88

1.0

1.5

Ratio of GNPI: DNA

2.0

0.00

0.05

0.10

0.15

0.20

0.4

Ratio of GNPII: DNA

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 5
6 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

J. Biomed. Nanotechnol. 11, 478485, 2015

Yao et al.

Gold Nanoparticles and Chemical Radiosensitivity

Counts (a.u.)

not lead to higher radiosensivity. A similar behavior is

an  of 33.97 mV are strongly anionic and expected
observed for thicker 10 ML films in which GNPs-II are
to be repelled from the phosphate groups of the DNA
more completely covered with DNA than in a 5 ML
molecule. In contrast, the nearly neutral GNP-I with an
film. In both cases, GNPs-II damage much less DNA
 of + 10.25 mV could be easily mixed with DNA in
compared to GNPs-I, with or without irradiation. Howsolution, becomes evenly distributed in the films of GNPever, all of the curves in Figure 3, with the exception of
DNA complexes and possibly binds to the phosphate group
the solution data, show a correlation between the results
of the DNA. The difference in  between the two GNPs
obtained with the irradiated and non-irradiated samples.
should therefore result in different binding states leadIn fact, damage increases when the films are bombarded
ing to a different sensitivity of DNA in contact with
by 10 eV electrons, but the line shapes of the curves in
the nanospheres. For this reason, we detected more DNA
Figure 3, for both the irradiated and non-irradiated samdamage when GNPs-I were in contact with DNA comples, remain essentially the same. This behavior seems
pared with GNPs-II, because GNPs-I have a stronger
to indicate that electron bombardment simply enhances
interaction with the DNA molecule. The stronger interacthe process, which causes SSB, when the films of DNAtion promotes additional DNA damage induced by LEE
GNP complexes are dehydrated under UHV. This process
impact (Fig. 3(A)), whereas the enhancement of damhas recently been investigated in detail by Huang et al.29
age from irradiation is almost negligible in the case of
It has been found that in the B form in a water soluGNPs-II.
tion, DNA is not sensitive to GNPs, whereas when in
Our hypothesis of the electrostatic potential interaction
the A form, either in vacuum or in solution, contact with
between GNPs and DNA is corroborated by XPS meaGNPs produces SSB. The amount of SSB increases with
surements of Au 4f spectra of the GNPs samples with and
the number of GNPs bound to DNA. Since during tranwithout DNA (Fig. 4). The Au 4f consist of the 5/2 and 7/2
scription the DNA-RNA duplexes adopt the A form, the
spin-orbit doublet peaks, with similar binding energies for
results of Huang et al. further showed that GNP could
GNPs-I and GNPs-II, as seen in Figure 4. In contrast, the
be genotoxic and that this toxicity could be cell-cycle
peaks of GNPs-I-DNA complexes are broader than those
dependent.29
of nude GNPs-I. Since no chemical bond exists between
In our experiments under UHV conditions, DNA in the
the GNPs and DNA, broadening of the bare GNPs-I peaks
GNP complexes is converted to the A form with only
indicates that the electrostatic interaction with DNA modby Publishing
to: Leon Sanche
structural water left. We thereforeDelivered
interpret our
results with Technology
ifies
Au2014
4f orbital
energies at the surface of the GNP.
IP: 132.210.107.48 On: Wed,
26the
Nov
17:12:23
no irradiation in terms of the interaction
of the GNPs
with Scientific
The GNPs-I-DNA
spectrum can be deconvoluted into two
Copyright:
American
Publishers
DNA. This interaction depends essentially on two key faccomponents. The one which has the same binding energy
tors: the positive charge on the GNP that binds it to DNA
as that of GNPs alone corresponds to the non-interacting
and the metallic and geometrical properties that catalyze
DNA damage. Binding of GNPs to DNA is expected to
locally bend the helix of the supercoiled configuration,
b
which can promote DNA damage in both the A and B
C
forms. However, the A form, compared to B form, has a
different, shorter and more compact helical structure. Thus,
GNP-II-DNA
more of the DNA phosphodiester backbone should be in
contact with the GNP in the A than in the B configuration. This characteristic alone could increase DNA damage
in the A form. More generally, different DNA configurations are known to have different sensitivities to different
A
physical and chemical environments.30, 31
GNP-II
The interaction between nude GNPs and DNA is a non32
specific electrostatic force. An effective parameter to
estimate the electrostatic potential at the electrical douGNP-I-DNA
ble layer surrounding a nanoparticle in solution is , the
electrostatic potential that exists at the shear plane of
a particle, which is related to both surface charge and
local environment. Nanoparticles with  lying between
GNP-I x1/6
10 and + 10 mV are considered approximately neutral,
81
84
87
90
93
while nanoparticles with a value greater than + 30 mV
Binding energy (eV)
or less than 30 mV are considered strongly cationic
or strongly anionic, respectively.33 Accordingly, GNPs-I
Figure 4. The Au 4f spectra of GNP-I, GNP-I-DNA (1:1), GNP-II
and II prepared in this study contain opposite interaction
and GNP-II-DNA (1:5), respectively. The Au 4f peaks of GNPcapability with negatively charged DNA. GNPs-II with
DNA are deconvoluted into pairs of components.
J. Biomed. Nanotechnol. 11, 478485, 2015

483

Gold Nanoparticles and Chemical Radiosensitivity

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
Delivered
by Publishing
LeontheSanche
the GNPsto:reach
cell nucleus they could be cell-cycle
sites. Thus, the existence of complex
binding
of GNPs-II Technology
IP: 132.210.107.48 On: Wed,
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

J. Biomed. Nanotechnol. 11, 478485, 2015

Yao et al.

Gold Nanoparticles and Chemical Radiosensitivity

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