nanoparticles promising future cancer therapy
Recently, the use of gold nanoparticles as potential selective tumor radiosensitizer has been
proposed as a major advance in radiotherapy. Experiments in living cells and in vivo have
demonstrated the effectiveness of metal nanoparticles when combined with low energy X-ray
radiation (below 1 MeV Linac conventional radiation). Other DNA studies have
performed in order to better understand the fundamental processes of awareness and
improving the method.
This paper proposes a new strategy based on a combination of platinum nanoparticles
irradiation with fast ions effectively used in hadron therapy. It notes in particular that
nanoparticles increase very lethal damage to DNA, with a factor close to 2 for efficiency
double strand breaks. To separate the effect of the nano-architecture design,
comparison with the effects of the dispersion of the metal atoms in the same concentration was
done. Therefore shown that nanoparticles awareness has been enhanced by the
self-amplified electronic cascades within the nanoparticles, which enhances the energy
deposit in the vicinity of the metal. Finally, the combination of fast-ion radiation
(hadron therapy) with platinum nanoparticles very should improve cancer treatment protocols.
Nanotechnology has taken a step forward in the
the fight against cancer . Recently, a combination of
radiation with nanoparticles has been proposed as a new
alternative to improve treatment protocols.
Radiotherapie conventional, based on X-rays and γ rays
radiation techniques are more widespread in the world
for the treatment of malignancies. These radiations
They have the advantage of penetrating the tissues, which allows
treating deep seated tumors. A major difficulty
is the lack of selectivity between the tumor and health
surrounding tissue. The application of these techniques
is therefore limited by the tolerance of normal tissues. The
challenge of radiation therapy, the future is to develop methods
to guide the dose deposition in tumors and improve the
The addition of a high-Z charged atom compounds
long ago proposed as a method to increase the effect of
ionizing radiation [2-5]. In a preclinical level, chemotherapy
with cis-platinum associated with X-ray radiation has been shown
encouraging results for the treatment of gliomas . The effect
of these compounds is well known at the molecular level in the
terms of rates of local amplification due to emission of electrons
around the Z atom high. This phenomenon has been
has been observed in different conditions of radiation [3, 7, 8].
The bottleneck of this innovative protocol is the low tissue
selectivity and high cytotoxicity of sensitizing agents.
Improvements in tumor targeting and awareness of efficiency are
Noble metals, especially gold, have been used in
Medicine . In recent years have seen impressive progress
in the applications of gold nanoparticles in particular. The
surface chemistry of nanoparticles opens up prospects for
targeting and differentiation of tissues by adsorption of
specific molecules. The optical properties of the infrared range
encourage the development of new techniques such as photodiagnostic
and photothermal therapy . More recently, gold
nanoparticles have been proposed as potential radiosensitizers
X-Ray for cancer therapy . Coated with gold nanoparticles
candidates with glucose, in particular, are good for treating breast
A thorough understanding of the processes induced by
nanoparticles in the genetic material is crucial to continue
awareness to improve the properties of these compounds. Hence,
DNA is used as a model system to quantify the effects of
ionizing radiation combined with nanoparticles in biological
conditions [13, 14]. Most studies have focused
on the effect of gold nanoparticles combined with x-rays.
However, it is worth noting that these studies,
have been conducted with laboratory X-ray of 200 keV
and down, the sources that are less relevant for physicians
applications [15-17]. In view of future applications
in cancer treatment, medical sources should be advanced
One of the most promising techniques in cancer treatment
is the hadron therapy (or proton therapy) in which carbon fast (or
Proton ions) are used as an alternative to hard X-rays . The
The advantage of these techniques stems from the unique ballistic
effect of ions, which differs markedly from the electrons and
photons . Furthermore, due to its large cross section of
interaction with matter, the ions are three times more efficient
conventional radiation. Finally, the fast-track treatment
ions opens new perspectives for an efficient and less traumatic
the eradication of cancer, including tumors resistant to radio sat
wrong in sensitive tissues inaccessible to surgery (brain,
eyes, tumors of children). This explains the rapid expansion of the
Hadron therapy and proton therapy centers in the world .
In order to improve the efficiency and targeting of medical
treatments, we propose to move in the development
protocols by combining nanoparticles of metal ions and fast
irradiation. This study focuses on the damage
physician-induced carbon ion beam loaded with DNA
platinum nanoparticles. Analysis of DNA damage using
to characterize the effect of this combination. To
separate the effects related to the nano-architecture design
A comparison with dispersed platinum atoms is also included.
This allows us to highlight the new properties of nanoparticles
are relevant to radiation biology. Finally, it is clear that the
Besides the high-Z nanoparticles with fast ion irradiation is a
very promising approach for future advances in cancer therapy.
2. Experimental Section
2.1. Preparation of samples
For simple and fast quantification of simple and complex
damage, DNA plasmid pBR322 (Euromedex) is used to
our study. It consists of a double-stranded supercoiled
4361 DNA base pairs (2.83 × 106 Da) diluted in
TE buffer (10 mmol L-1 Tris-HCl (pH 7.6) and
1 mmol L -1 ethylenediaminetetraacetic acid (EDTA)). Plasmid
DNA has the advantage of having three conformations,
conformations know supercoiled, circular and linear, when,
, Respectively, without interruption, a break in a chain, two breaks
into two strands (separated by less than ten base pairs) are
produced in the DNA molecule. The three conformations
are separated by migration in agarose gels
subjected to an electric field (10 cm V-1, 70 mA). Before
irradiation, DNA samples containing more than 95% supercoiled,
5% of circular and not linearly.
Chlorine platinum 2.2?: 6?
, 2?? terpyridine (PTTC) is
a commercial product (Sigma Aldrich Chemie GmbH,
Schnelldorf, Germany). Dilute in pure water, in a
concentration of 4.23 × 10-5 mol L-1 determined by the absorption of
spectroscopy (ε = 25 100 mol cm-2 to λ = 278 nm, 1.06 OD).
The solution was used without further purification.
Nanoparticles of platinum (PtNP) coated with polyacrylic
acid (PAA) (Sigma Aldrich) were synthesized by radiolysis
the reduction of platinum complexes (Pt (NH3) 4Cl2, H2O) (Sigma
Aldrich) (10-4 mol L-1) in aqueous solution containing or
not containing PAA (10-2 mol L-1). The solutions are
deaerated before irradiation by bubbling nitrogen.
Irradiation was carried out in a panoramic source (60Co
source) at a dose rate of 2.2 kGy h-1 (1 Gy = 1 J kg-1). The
radiation dose was about 1000 Gy. Irradiated solutions
were protected from light and stored at 4 ◦ C. Transmission
electron microscopy (TEM) observations were made in
a JEOL JEM 100 CXII transmission electron microscope
an accelerating voltage of 100 kV. The solutions eventually
diluted to a concentration of metal atoms of 4.23 ×
10.5 l mol-1, similar to the concentration reached with PTTC.
The nanoparticles were finally kept in a buffer composed
ion of chlorine and ammonia to 4.23 × 10-5 mol L-1. It
is a crucial aspect to consider in this type of experiment
because chlorine ions as anions strongly modify the
effects of radiation and sensitization in biological systems. In
this work, the concentration of ions remaining in the samples of
after addition of DNA solutions are much lower than the
concentrations of ions in the Tris-EDTA buffer for DNA, and
should therefore not induce any artifact.
DNA samples radiosensitizer complexes are
prepared as follows. 18 ¶ l aliquots each containing 500 ng
DNA (1 microliter). In order to maintain constant concentration of
counter-ions, 12.3 ¶ l of TE buffer was added to each
sample. Radiosensitizers were added to the samples, so that
concentrations of platinum atoms remain constant when
PTTC and PtNPs were used (2.4 microliter). The final volume was
adjusted to 18 ¶ l of pure water. The DNA was incubated
with radiosensitizer for 1 h before irradiation.
It is widely accepted that the positive charge PTTC
is electrostatically bound to DNA (negatively charged) .
Additional studies have shown that for so many PTTC,
an insignificant fraction of the PTTC is free in solution and a
PTTC complete DNA binding was observed .
Platinum nanoparticles are prepared from precursors
containing ammonium groups. We can expect the ammonia groups
adhere to the metal nanoparticles. Therefore, given to groups
a positive charge allowing binding to DNA as
proposed in Figure 1.
The complexes are prepared so that they contain in
average of one platinum atom per seven to eight base pairs
average (an atom of platinum per 15 atoms of phosphate, 500
platinum atoms plasmid), as used elsewhere [3, 8].
The nanoparticles are added to DNA with the same number of
plasmid platinum atoms, which corresponds to a final
PtNP: about DNA nanoparticles by a ratio of two plasmids
(3 nm in diameter, 1000 atom per PtNP).
The addition of either had no radiosensitizing
harmful effects on DNA, as shown in a systematic way
Irradiation for C6 ions were performed at the Heavy Ion
Medical Accelerator (HIMAC, Chiba, Japan), one of the most
Advanced therapy centers in the world Hadron. The beam
set at an energy of 276 MeV amu-1, which corresponds to a
linear energy transfer (LET) in the middle of 13.4 keV microns-1
the location of the sample, with a dose rate of approximately
4 min Gy-1.
DNA solutions were placed in cups Eppendorfs.
The thickness of the irradiated samples was 1.5 mm, ensuring
a statement of constant dose along the path through the sample.
Irradiation was carried out under atmospheric conditions at
The doses ranged from 0 to 360 Gy, which corresponds
irradiation time to a maximum of about 50 min.
Samples of 18 Pl was divided into two aliquots for
to preserve half of the proceeds in case of artifacts in the
analysis. 9 ¶ l samples were loaded with 1 ¶ 6 × loading
dye solution. Electrophoresis was performed in 1.7%
agarose gel in an electric field of 10 cm V-1, at 4 ◦ C
3.5 hours After migration, the gel was stained with ethidium
bromide (1 mg ml-1), and lines of DNA revealed in
ultraviolet (UV) light (302 nm) and recorded by a CCD camera.
Image analysis software (Image Quant) was used to quantify
intensity of lines of DNA in different DNA conformations.
The supercoiled plasmid (S) to bind to 1.47 times less ethidium
bromide relaxed (R) and linear (L) conformations. The
performance of DNA damage was determined as follows:
Total = 1.47 × S + R + L R? = R / total
S? = 1.47 × S / L total? = L / total.
The number of fragments of individual strands and double strand breaks
by the plasmid were determined by the respective following
Performance of SSB (breaks per plasmid) = ln
1 – L?
Yield OSD (breaks per plasmid) = L?
1 – L? .
No significant artifacts due to the union of PTTC or NP for
DNA is found on electrophoresis. The presence of
radiosensitizer does not change the conformation of DNA,
as shown TEM images also in the case of DNA PTTC
complex (not shown here). This result coincides with the
hypothesis that is proposed here radiosensitizers bind to DNA
by non-covalent electrostatic interaction.
3.1. Synthesis and characterization of platinum nanoparticles
The synthesis of platinum nanoparticles was mediated by
γ-ray radiolysis of water. Hydrated electrons and
Figure 2. TEM of platinum nanoparticles coated with PAA
synthesized by radiolysis, at 2.2 kGy h-1.
Figure 3. Average number of single strand breaks (SSB) by
plasmid versus dose of pure DNA (?), DNA in the presence of
PTTC (?) In the presence of DNA-coated PtNP PAA (•) and
the same as the last case added in DMSO solution (◦).
The samples were irradiated by ions C6 + 276 MeV amu-1
(LET = 13.4 keV microns-1).
reducing radicals (H ·) produced during water radiolysis induced
reduction of homogeneous nucleation . The main
advantage of this method is to produce nanoparticles in a
final solvent whose chemical composition (mainly NaCl) is
supports the use of biological systems and study
the effects of radiation (eg, alcohol should be prohibited
due to its radical capturing properties). Platinum
PAA-stabilized nanoparticles are 3 nm in average diameter
(Figure 2), which corresponds roughly to 1000 platinum
atoms in a particle. TEM measurements show that coated
nanoparticles stored eight days in a dark room at 4 ◦ C remain
3.2. The radiation-induced DNA damage
The effect of platinum is characterized by the quantifying the
DNA damage, single strand breaks (SSB) and double-stranded
breaks (DSBs), induced by irradiation. The results
with DNA loaded with platinum nanoparticles and irradiated
Figure 4. Average number of double strand breaks (DSBs) by
plasmid versus dose of pure DNA (?), DNA in the presence of
PTTC (?) And DNA in the presence of PAA PtNP coated (•)
and the same as the last case added in DMSO solution (◦).
The samples were irradiated by ions C6 + 276 MeV amu-1
(LET = 13.4 keV microns-1).
Table 1. Yields of SSB and DSB (slopes m) induced by
C6 + 276 MeV amu-1 naked DNA plasmid and plasmids loaded
with PTTC PtNP coated or PAA.
m (SSB) (skipping factor Awareness
m (OSD) (skipping
(× 10-11) SSB DSB
Pure DNA, 19 (± 1) 3.0 (± 0.5)
PTTC DNA + 31 (± 1) 4.9 (± 0.6) 1.63 1.63
DNA + PtNPs 26 (± 3) 6.5 (± 0.8) 1.37 2.17
by carbon ions of 276 MeV amu-1 (energy deposition in the
13 microns keV-1), are presented in Figures 3 and 4 for a dose
irradiation ranging from 0 to 200 Gy. The effect of
platinum nanoparticles is compared with the effect of platinum
atoms (PTTC) at the same concentration of atoms, well known
previous studies . Pure DNA is used as a reference.
Experiments with pure DNA, DNA-loaded
PtNP with PTTC and show that the number of DNA breaks
increases linearly with dose. When platinum
nanoparticles are added to DNA or atoms-radiation
effects are amplified. The results obtained with PTTC
are in good agreement with previous work .
Yields of SSB and DSBs is defined as the number of
breaks induced by gray and Dalton. They correspond to the
slopes of the dose-response curves, divided by the molecular
Da weight plasmid (2.83 × 106 per plasmid to pBR322).
The slopes (m) are reported in Table 1.
The values of SSB and DSB yields illustrate the amplification
effect induced by platinum compounds, where carbon ions
are used as ionizing radiation. These results confirm the sensitization
the properties of metal nanoparticles [13, 14]. Given
of SSB, the effect PtNP amplification is similar to the effects of
observed by other groups [13, 14]. In particular, the SSB
loaded with DNA yield PtNP (26 x 10.10 breaks per gray
by Dalton) is the same order of magnitude as that obtained
by Butterworth and others when 5 nm diameter gold nanoparticles
were combined with X-rays . However, the effect on the complex
damage (OSD) is clearly superior in this experiment.
More interesting, at the same concentration of platinum,
the ratio of SSB to DSB yields are lower in the presence of
nanoparticles (43) that the scattered atoms (63). Breaks
therefore more complex with the nanoparticles. Therefore, the
but also the quality of amplification of radiation is damage
closely related to the design of nano-sensitizers. In
in particular, the addition of platinum nanoparticles improves
strongly the lethality of the damage and therefore the biological
A simple characterization of the sensitizers is offered
by factors of awareness, defined as the ratio of SSB
(respectively DSB) of performance in DNA loaded with platinum
compound on the SSB (DSB respectively) obtained performance
pure DNA (see Table 1, the last column). The awareness factor
of PTTC is about 1.6 per SSB and DSBs. For PtNP, the
awareness respective factors are 1.4 for the induction of SSB
and around 2.1 for the induction of the DSB. In the work of
Butterworth et al with gold nanoparticles combined with X-rays,
an amplification factor of 2 is reported by SSB and
only 1.2 per DSBs. Finally, the protocol is the
combination of platinum nanoparticles 3 nm in diameter rapidly
ion irradiation is, thus far, the most efficient way to induce lethal
The interaction of ionizing particles (charged particles,
photons) with the biological material is described in detail in the
review Mozumder and Hatano . In short, the ionization of the
water molecules and the production of secondary electrons
along the main track are considered as the main processes
occurring in the initial phase (t <s 10-12) (see Figure 5).
As a result, the uneven distribution of electrons
(Blubs and spurs) appears along the track. Before salvation,
low-energy electrons and high interact with their environment
and could ionize the water or biological molecules.
In the presence of metal compounds, additional
ionizations take place because of the high-ionization cross
section of high Z atoms . Incident ionizing particles
(such as ions) and secondary electrons produced along the track
efficient can excite the inner and outer shells of the metal.
Ionizations in inner shells, in particular, are followed by Auger
excitation processes, resulting in amplification of the
the emission of electrons from the metal. Approximately ten
Auger electrons are emitted after the ionization of the platinum
L-shell. In short, the presence of high-Z atoms in the
locally amplified half ionization density and dose
When DNA is loaded with platinum, Auger electrons can
interact directly with DNA and induce breaks in the chain
(direct effect). Which otherwise interact with the surrounding water
molecules to produce radical groups can continue
DNA damage (indirect effect). The role of water radicals is
investigated by the addition of a debugger radical (dimethylsulfoxide)
in some experiments (see Section 2). In the case of pure
DNA and DNA loaded with platinum, the
SSB induction of DSBs and is very close (see
Figures 3 and 4). This result confirms that the induction of DNA
damage and, more interestingly, the amplification of radiation
effects due to the metal are mostly related to production
radicals of water near the metal [7, 14]. Align processes
(unmediated by solvent molecules), such as the interaction of
electrons and secondary ions with DNA, can be considered as
minor contributions [23-25].
As explained above, the presence of metal changes locally
dose deposition. Therefore, in relation to the ionization cross
section of the metal and depends on its atomic mass (Z).
To explain the observed difference between platinum
atoms and platinum nanoparticles, additional mechanisms
architectures for nano-specific design must be invoked.
In the nanoparticles, the atoms are closely linked. Therefore,
electrons emitted from an atom of platinum and Auger electron
the valence electrons can excite the surrounding metal atoms. As
Consequently, the advantage of metal nanoparticles is due
of self-amplifying cascades generated electronic
within the nanoparticles leading to a net improvement
emission of electrons and radical production of water. This
strong disturbance that is induced at the nanometer scale
volume around the nanoparticles, explains the relative increase
lethal damage (DSBs) compared with SSB. In fact DSBs,
that require two bonds separated by pauses of a few nanometers
(the two DNA strands are separated by 2 nm) are strongly
catalyzed by nanoparticles. By contrast, SSB, which
require only single events to break bonds, decreased
presence of nanoparticles, due to reduced number of
platinum sites in DNA.
After the emission of electrons, platinum nanoparticles are left
with very positive charges in the middle. Therefore
multiple charge transfer molecules surrounding
positively charged nanoparticles can efficiently contribute to the
ionization of water molecules surrounding it and therefore
radical production in the vicinity.
Calculations are in progress in order to quantify the
contributions of different routes.
We present here the first study that highlights the outstanding
sensitizing properties of platinum nanoparticles in comparison with
to the metal atoms. Effects on DNA were investigated in
to compare and optimize the biological effectiveness
sensitizers in biological conditions. The fast carbon irradiation
ions is considered in this paper to review applications
in one of the most promising techniques for cancer treatments,
Our main result is that the platinum nanoparticles improve
strongly biological efficacy of radiation. The nanodesign
the architecture of the particles plays a crucial role.
Sensitization properties of nanoparticles are attributed to
specific self-amplified electronic cascades, as well as
the charge transfer processes. The rapid spread of these effects
in a nanoscale volume explains the amplification of the
lethal damage in DNA.
Finally, this work shows that the combination of platinum
nanoparticles with fast ions opens new perspectives in
cancer therapy. Further optimizations are underway in
to combine awareness and orientation of the properties of
nanoparticles, together with the ballistic effects of ions.
The authors acknowledge Patricia Beaunier Laboratory
Surface R’eactivit’e, Universit’e Paris VI, TEM, high
Resolution TEM (HRTEM) observations.
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