METHODS
The effects of different concentrations of AuNPs (0.75 ?g/ml, 0.5 ?g/ml, 0.25 ?g/ml, and only medium)
and 2 Gy ionizing radiation (IR) were investigated on the L929 fibroblast, DLD-1 colon, and H1299 lung
cancer cell lines. Cytoplasmic and nuclear membranes treated with hematoxylin and eosin and double
staining were evaluated with light and fluorescence microscopy. Cytotoxicity was determined with the
WST-1 method.
RESULTS
All particles were spherical in shape with 60.98 nm in size. Cell surviving ratios without AuNPs were
96.34% in L929, 89.68% in DLD-1, and 76.93% in H1299 for a single 2-Gy radiation. These ratios were
94.2%, 62.58%, and 40.52% for L929 cells; 72.70%, 41.15%, and 26.71% for DLD-1 cells; and 34.72%,
28.27%, and 17.84% for H1299 cells at concentrations of 0.25 ?g/ml, 0.5 ?g/ml, and 0.75 ?g/ml AuNPs,
respectively.
CONCLUSION
At increased concentrations, isolated unwanted cytotoxic effects of AuNPs could be observed. Radiosensitizing
effect of PEI-coated AuNPs depends on cell type and AuNP concentration.
Keywords: Gold nanoparticle; megavoltage; polyethyleneimine; radiosensitization
Nanotechnology is a rapidly developing branch of
science. Its use in radiation oncology has three major
aspects: 1) facilitate image-guided radiotherapy (IGRT)
through mechanisms based on increased uptake of
particles by cancer cells similar to positron emission
tomography imaging [
Gold nanoparticles (AuNPs) are usually various
shaped with a size of 1?100 nm and a gold core covered
by surface coating.[
Over the past decade, the field of AuNP-based radiosensitization
has undergone a tremendous growth;
this fact is demonstrated by the exponential increase
in the number of publications on this topic. Studies
regarding AuNP-mediated radiation enhancement
have been done from the early 2000s.[
This study aimed to characterize the cytotoxicity
on normal fibroblasts (L929), colon (DLD-1), and lung
(H1299) cancer cell lines via AuNP-mediated radiation
dose enhancement under MV energies.
AuNP Synthesis
Irradiation Setup
The gafchromic film was placed under the dishes to
evaluate dose homogeneity and to irradiate with a single
dose of 2 Gy. After radiation exposure, the dosimetry
film demonstrated that the target area was homogeneously
exposed to radiation.
Hematoxylin-Eosin Staining
Double Staining
WST-1 Cytotoxicity Test
Following incubation, the absorbance was measured
in a microplate reader at a wavelength of 440 nm
to determine the toxicity of the gold particles alone.
In the second phase of the experiment, after 24 h
exposure with AuNPs, cells were irradiated with 2 Gy
photon beams. After irradiation, AuNP-exposed and
unexposed cells (control) were incubated in culture
medium for another 24 h, and then viability was tested
for the IR and IR+AuNP groups.
Radiation Enhancement Ratio
AuNPs coated with polyethyleneimine (PEI) and
3-aminophenyl boronic acid, which were previously
synthesized for another study, were used in our work.
[
Megavoltage X-ray (6 MV) irradiations were performed
using the Elekta linear accelerator. Petri dishes
contain a layer of malignant cells plus about 2 mm of
medium and 18 mm air. The prescribed dose was 2 Gy,
and SSD was set to 100 cm using a 20×20 cm field. Six
centimeters of plexyglass material (water-equivalent)
was placed on top of the dish. The dose rate was 3.55
Gy/min.
A 20×103 cells/well of L929, DLD-1, and H1299 cells
were placed in a 48-well plate of 10% fetal bovine serum
and 1% penicillin streptomycin containing DMEM and
RPMI and incubated with 5% CO2 at 37°C for 24 h.
Afterwards, cells were treated with different amounts
of AuNPs (0.25 ?g/ml, 0.5 ?g/ml, 0.75 ?g/ml) and incubated
another 24 h and then 2 Gy RT was applied.
Alcohol was put into cells in decreasing concentrations
(100%, 96%, 80%, 70%, 50%) for 1 min for dehydration.
Cell morphology was examined under the light
microscope. Experiments were repeated thrice.
Double staining was performed to quantify the number
of apoptotic and necrotic cells. The L929, DLD-1,
and H1299 cells were placed in 10 ml of PBS solution
in a 48-well plate (20×103 cells/well). Ribonuclease A
inhibits cytoplasmic RNA staining, and propodium
iodide stains necrotic and Hoescht 33342 stains apoptotic
cells. Cells were subjected to RPMI and DMEM
and incubated with 5% CO2 at 37°C for 24 h. Then
three different concentrations (0.75 ?g/ml, 0.5 ?g/ml,
0.25 ?g/ml) of AuNPs were added and incubated for
another 24-h repeated thrice. Afterwards, they were
washed with PBS (after the incubation, the cells media
were discarded) and stained with 70 ?l double staining
dye for 15 min at dark. Cells were examined with
FITC- and DAPI-filtered fluorescent inverted microscope.
A 20-fold magnification was used for images.
Initially cells were plated for 24 h, and then treated
with 0.25 ?g/ml, 0.50 ?g/ml, and 0.75 ?g/ml 60.98 nm
AuNPs for 24 h. Then cells were washed; and cell media
was discarded and incubated in fresh culture medium
without 100 ?l phenol red. The cells were then exposed
to 10 ?l WST-1 solution for 4 h, and plates were read
using a microplate reader at 440 nm. The cell numbers
in the control and AuNP-exposed samples were
counted; thus, toxicity of different concentration of
AuNPs was analyzed.
The radiation enhancement ratio (RER) is defined as
the ratio of survival fractions without and with AuNPs
for a specific dose that is 2 Gy in this setting. It has
the benefit of directly comparing the biologic response
caused by the nanoparticles at a specific dose level.
Cells were aggregated, lost their cytoplasmic membrane integrity, decreased in volume, and detached from the plate. These changes were most abundant in H1299 cells and least in fibroblasts and exacerbated with IR. Cross-linking between positively charged AuNPs with glycosaminoglycan and collagens caused intercellular organic material accumulation leading to cytoplasmic membrane damage. This may result in decreased uptake of nutrients in cancer cells and thus cell death.
Double Staining of Nucleus
The PI-stained necrotic cell ratios and microscopic images
are presented in Table
Viability
Cell surviving ratios without AuNPs were 96.34% in L
929 (Fig.
Radiation Enhancement Ratio
Radiation enhancement ratio (RER2Gy) for DLD-1
was 1.23 and for H1299 was 2.21 with 0.25 ?g/ml concentrations
of AuNPs.
In this study, we used 60.98 nm spherical AuNPs
coated with cationic charged PEI and 0.25 ?g/ml
AuNPs concentration considered to be safe so that at
least 69% viability observed cancerous cell lines and fibroblast continued to proliferate. The toxic effect due
to AuNPs showed a significant difference between normal
and cancerous cells at low concentrations, but this
difference gradually diminished with increasing concentrations.
The level of toxicity was also different for
different tumor types; H1299 appears to be the most
vulnerable group. In a study by Coulter et al., they
found that AuNP uptake was preferentially observed
in tumor cells, and cytotoxicity was low, so that particle
concentration causing 50% of growth inhibition
in cell culture defined as IC50 for normal cells were 14
times higher than tumor cells.[
Formerly, radiosensitization has mainly been attributed
to physical dose enhancement occurring at kilovoltage
(kV) photon energies by means of photoelectric
absorption of gold as a high Z material. However,
data about different mechanisms of radiobiological effects
regarding radiosensitization are growing [
The surface coating is an important parameter to
control particle stability solubility and biocompatibility.[
Chithrani et al. evaluated the size and shape dependence
of AuNPs" uptake into mammalian cells,
and observed the highest uptake and radiosensitization
with approximately 50 nm compared to the 14 nm
and 70 nm.[
Wang et al. reported the radiosensitization effect
of glucose-capped AuNPs with different sizes (16 nm
and 49 nm) on triple-negative breast cancer cells in
the presence of MV energy. They showed increased
RS effect with 49 nm AuNPs with an SER of 1.86 compared
to 16 nm AuNPs with an SER of 1.49. Moreover,
they demonstrated that the rate of the cells arrested in
G2/M phase were increased with AuNPs" exposure,
more with 49 nm AuNPs, a possible mechanism for
enhanced radiation sensitization effect because G2/M
phase is the most radiosensitive phase of the cell cycle.
[
There are certain limitations of the study. First,
although staining of cells showed us cell membrane,
cytoplasm, and nuclear damage, we were not able to
determine the intracellular distribution of AuNPs, in
terms of uptake and localization that can affect both
the isolated toxicity of AuNPs and radio sensitization.
Defining the underlying mechanism for RS effect is
also critical for possible further clinical application.
AuNP-related cytotoxicity was observed not only
in cancerous cells but also in noncancerous fibroblasts.
This toxic effect is more pronounced at increasing concentrations
that can limit the use of AuNP. This study
also suggests that lower concentration of AuNPs acting
only on cancer cells can be used as RSs and are celltype
dependent.
Peer-review: Externally peer-reviewed.
Conflict of Interest: No conflict of interest.
Financial Support: No financial support.
Authorship contributions: Concept - S.A.A., M.T.; Design
- S.A.A., M.T.; Supervision - S.A.A., M.T.; Materials -
S.A.A., M.T.; Data collection &/or processing ? S.A.A., M.T.;
Analysis and/or interpretation - S.A.A., M.T.; Literature
search - S.A.A.; Writing - S.A.A.; Critical review - S.A.A.
