Biological mechanisms of gold nanoparticle radiosensitization
Rosa et al. Cancer Nano
Biological mechanisms of gold nanoparticle radiosensitization
Soraia Rosa 0
Chris Connolly 0 1
Giuseppe Schettino 1
Karl T. Butterworth 0
Kevin M. Prise 0
0 Centre for Cancer Research and Cell Biology, Queens University Belfast , 97 Lisburn Road, Belfast BT9 7AE, Northern Ireland , UK
1 National Physical Laboratory , Teddington, London TW11 0LW , UK
There has been growing interest in the use of nanomaterials for a range of biomedical applications over the last number of years. In particular, gold nanoparticles (GNPs) possess a number of unique properties that make them ideal candidates as radiosensitizers on the basis of their strong photoelectric absorption coefficient and ease of synthesis. However, despite promising preclinical evidence in vitro supported by a limited amount of in vivo experiments, along with advances in mechanistic understanding, GNPs have not yet translated into the clinic. This may be due to disparity between predicted levels of radiosensitization based on physical action, observed biological response and an incomplete mechanistic understanding, alongside current experimental limitations. This paper provides a review of the current state of the field, highlighting the potential underlying biological mechanisms in GNP radiosensitization and examining the barriers to clinical translation.
Cancer therapy; Radiation therapy; Radiosensitization; Gold nanoparticle
Radiation therapy is frequently used in the treatment of cancer, with both curative and
palliative intent. However, radiation doses that can be delivered to patients are limited
by toxicity in the surrounding healthy tissue. Many efforts in Radiation Oncology have
focussed on approaches that aim to preferentially sensitize tumours to radiation whilst
minimizing effects in normal tissues. An approach to maximize the differential response
between tumour and normal tissue response, termed therapeutic ratio, is through the
promising radiosensitizer in this regard due to its high atomic number and mass energy
coefficient relative to soft tissue. As shown in Fig. 1, the mass energy coefficient of gold is
100–150 times greater than that of soft tissue in the keV energy range (Hubbell and
Seltzer 1996). Consequently, there is an increased probability of photoelectric interaction at
lower energy levels, resulting in increased energy deposition at the target site. However,
considering the depth dose limitations of keV X-rays, MV energies are used as the
clinical standard for external beam radiotherapy. At these energies, significant
radiosensitization would not be expected based on the ratio of mass energy absorption coefficients
of gold and soft tissue.
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Fig. 1 Photon mass energy absorption coefficients of soft tissue and gold. The ratio of the mass energy
absorption coefficients is shown as a function of photon energy (Hubbell and Seltzer 1996)
Despite this, observed experimental findings deviate from predicted levels of
radiosensitization, with effects observed at concentrations lower than predicted. Figure 2 shows
the X-ray dose modification results against the predicted degree of dose enhancement,
based on the gold concentrations and X-ray energies used for a range of experimental
studies (Butterworth et al. 2012). The observed degree of radiosensitization in almost all
of the experimental results is much greater than the predicted increase in physical dose.
Physical dose variation between differing nanoparticle preparations and cell lines can be
seen to be much smaller than predicted, highlighting this as an unknown in cellular
radiobiological response which is not driven by increasing the total dose delivered to the cells
behind the radiosensitizing properties of gold nanoparticles (Butterworth et al. 2013).
Fig. 2 Comparison of predicted and observed values of dose enhancement for gold nanoparticles at both
megavoltage and kilovoltage energies. “Increase in physical dose” here refers to the ratio of the additional
dose deposited by X-rays in the system due to the addition of GNPs to that which would be deposited in
the absence of gold. The observed data in this figure are dose modification results from in vitro experiments,
while the predicted dose increase is based on the gold concentrations and X-ray energies used. The dashed
line shows the trend which would be followed if the sensitizer enhancement ratio directly followed predicted
increases in physical dose (Butterworth et al. 2013)
The ISO international standard defines nanoparticles as ‘particles which typically do
not exceed 100 nm in any of their dimensions’ (Michael et al. 2005). GNPs consist of
a gold core which can be generated from various synthesis techniques to give rise to a
wide range of different sizes and shapes, covered by a surface coating (Grzelczak et al.
2008). The surface coating can be functionalized for several uses, such as imaging,
delivery and diagnostics (Rana et al. 2012; Ghosh et al. 2008; Mieszawska et al. 2013; Sperling
et al. 2008).
This review follows on from our 2012 paper “Physical basis and biological
mechanisms of gold nanoparticle radiosensitization” and aims to review the current state of
the field. This will be accomplished by addressing three main points: the physical basis of
GNP radiosensitization; the biological mechanisms of GNP radiosensitization; and the
uptake, imaging potential and toxicity of GNPs in biological systems.
Physical basis of GNP radiosensitization
It is known that ionizing radiation can directly or indirectly damage DNA and disrupt
the atomic structure of other biomolecules (Kavanagh et al. 2013; Azzam et al. 2012).
DNA repair mechanisms may fail leading cells to stop dividing, die or be mis-repaired,
thus acquiring mutations that can result in malignant transformation (Begg et al. 2011).
Therefore, avoiding normal tissue is of significant importance in reducing secondary
side effects of radiotherapy.
However, one of the major challenges of radiotherapy is its lack of selectivity due to
the similar mass energy absorption properties of both cancer and healthy tissues
(Butterworth et al. 2012). In order to overcome this, agents such as metal-based
nanoparticles (with high Z) have been found to improve the contrast between tumour and soft
tissues, thus presenting radiosensitizing properties and potentially improving tumour
control, reducing side effects and increasing survival when compared to radiotherapy
alone (Herold et al. 2000; Regulla et al. 2002). Those absorb more energy per unit mass
than soft tissue increasing the local dose deposited in the tumour (Hubbell and Seltzer
The main physical mechanisms through which radiation interacts with nanoparticles
in the keV range are the Compton and Photoelectric effects, where an incident photon
can either be partially or fully absorbed by an electron from the atom, causing its
ejection (McMahon et al. 2016). The Photoelectric effect is a competing process in which
the electrons are ejected preferentially from an inner atomic orbital. The vacancy left
can then be filled by another outer shell electron that falls to its place, further releasing
low-energy photons promoting a cascade release of secondary electrons (Butterworth
et al. 2012). This process is called the Auger cascade and it is the major contribution to
the production of low-energy electrons that have a range of few micrometres and cause
highly localized ionizing events (Fig. 3) (Butterworth et al. 2012; Xie et al. 2015).
High-Z elements such as iodine, gadolinium and gold have been shown to have the
ability to image and radiosensitize tumours (Herold et al. 2000; Regulla et al. 2002;
Luchette et al. 2014; Martin 2002). Furthermore, gold has been shown to be
biocompatible which makes it an ideal candidate as a radiosensitizer (Shukla et al. 2005; Hainfeld
et al. 2010; Hainfeld et al. 2004).
Fig. 3 Schematic illustration of the photoelectric, Compton and Auger Effects. The Compton effect is
represented in blue, the photoelectric effect in green and the Auger effect in red as described above
GNPs were initially thought to enhance radiosensitization through physical processes
but extensive data have shown chemical and biological components involved in the
radiosensitization process as there is a poor correlation between experimental biological
results and dosimetric calculations (Butterworth et al. 2012; Ionita et al. 2005; Jain et al.
Simulations suggest that the presence of 1% of gold could double the dose deposited
using X-rays at keV energies and experimental evidence has demonstrated their ability to
radiosensitize (Cho 2005). On the other hand, theoretical calculations predict that using
MV X-ray sources there would be no significant increase in the dose deposited
(Butterworth et al. 2012, 2013). Recently, it has been demonstrated by Monte Carlo
simulations that there is an increase in secondary electron production when gold is irradiated
with X-rays at 6 MV compared to water (Ka 2015). Also, in vitro and in vivo
experimental evidence demonstrates a higher radiosensitization effect compared to the predicted
increase in physical dose expected, in this energy range, suggesting a strong biological
component in the radiosensitization process as shown in Fig. 2.
Biological mechanisms of GNP radiosensitization
The main mechanisms identified as being involved in the biological response of cells
to gold nanoparticle radiosensitization are the production of ROS and oxidative stress,
DNA damage induction, cell cycle effects and potential interference with the bystander
effects (Fig. 4) as described in the following sections in more detail.
ROS and oxidative stress
Gold is believed to be chemically inert. However, growing evidence suggests that their
surface is electronically active, thus catalysing chemical reactions and promoting an
increase in the production of ROS (Ionita et al. 2005; Mikami et al. 2012; Ionita et al.
Fig. 4 Schematic representation of the biological mechanisms involved in GNP radiosensitization. GNPs
influence oxidative stress, DNA damage, cell cycle and bystander effects
2007; Zhang et al. 2003). This seems to be more evident in small NPs (nanoparticles)
<5 nm in diameter that present a greater surface area-to-volume ratio (Li 2006; Hvolbaek
et al. 2007; Cheng et al. 2012). One of the identified mechanisms as a possible reason for
cytotoxicity is through the interaction of the NP surface with O2. In this process, donor
electrons are transferred from the surface of the NPs to oxygen molecules generating
superoxide, which can lead to ROS production through dismutation. This has been
identified for single-component materials and for transition metals on the nanoparticle
surface, such as Fe and vanadium, which take part in the formation of active sites (Li 2006).
In addition to reactive radicals on the NP surface, there are other sources of
oxidative stress such as the redox groups in the coating, contaminants from the production
method of non-metal NPs and oxidant-inducing properties of NPs (Li 2006; Fard et al.
2015; Tournebize et al. 2012). Oxidative stress causes damage to cell membranes, DNA
and protein being identified so far as one of the major causes of NP cytotoxicity (Pan
et al. 2009; Xia et al. 2006). Several reports of ROS production and oxidative stress
induced by nanoparticles alone have been published as well as in combination with
ionizing radiation as discussed below.
Nanoparticle‑induced oxidative stress
In vitro reports have shown enhanced ROS production in the presence of GNPs and
the absence of radiation by various groups (Li 2006; Pan et al. 2009; Coulter et al. 2012;
Wahab et al. 2014; Chompoosor et al. 2010; Tang et al. 2015; Mateo et al. 2014).
Mitochondria seem to play a role in it with data indicating loss of function due to high
intracellular ROS levels. ROS can oxidize the mitochondrial membrane disrupting its
potential and leaking more superoxide anions into the cytosol which can in turn be
converted into H2O2 molecules. These further diffuse across membranes and damage DNA
(Hei 2015; Havaki et al. 2015).
This is supported by experimental findings using 1.4-nm triphenyl monosulfonate
(TPPMS)-coated GNPs that promote loss of mitochondrial potential through elevated
oxidative stress causing necrotic cell death (Pan et al. 2009). Moreover, it was also found
that antioxidants containing thiol groups bind to the surface of GNPs. This suggests that
GNPs can bind these antioxidants inside cells inhibiting endogenous reducing agents
from acting and therefore reduce the redox capacity of the cells (Pan et al. 2009). GNPs
with different sizes, shapes and surface properties can also promote apoptosis or
necrosis through ROS generation (Coulter et al. 2012; Wahab et al. 2014; Chompoosor et al.
2010; Tang et al. 2015; Mateo et al. 2014). Tiopronin-coated GNPs led to necrosis by
enhanced ROS production after 24-h exposure in HeLa and L929 (fibroblast) cells (Li
2006). Also, dose dependency has been found to increase ROS production with
citrate GNPs. These lead to apoptosis due to mitochondrial dysfunction with associated
upregulation of caspase 3 and 7 (Wahab et al. 2014). Furthermore, mitochondrial
membrane polarization is decreased and mitochondrial oxidation is increased when cells are
exposed to GNPs (AuroVistTM) (Taggart et al. 2014). Moreover, in an indirect way, Au
(gold) clusters (Au25peptide9) have been shown to dramatically increase ROS
production via inhibition of thioredoxin reductase 1 (TrxR1) activity (Liu et al. 2014). TrxR1 is a
regulator of redox reactions within cells and its binding to the GNP surface dramatically
increased ROS levels inducing apoptosis (Arnér and Holmgren 2000; Fang et al. 2005;
Tonissen and Trapani 2009; Omata et al. 2006).
In combination with ionizing radiation, GNPs contribute to an increased
radiosensitization through which enhanced radical production has been observed following
irradiation in the presence of glucose-capped GNPs with 90 kVp and 6 MV X-rays (Geng
et al. 2011). Also, GNPs in water exposed to 100 kVp X-rays can promote an increase
in hydroxyl radicals (1.46 fold) and superoxide anions (7.68-fold) (Misawa and
Takahashi 2011). These increased levels of ROS and oxidative stress can trigger apoptosis as
observed with 14-nm particles, in ovarian cancer cells, exposed to a MV and kV X-ray
source (Geng et al. 2011).
Taggart et al. (2016) established a biological mechanism significantly contributing to
radiosensitization, using 1.9-nm thiol-coated GNPs. Irradiation in the presence of GNPs
led to an interaction between GNPs and the cell membrane protein disulfide
isomerase (PDI), resulting in the disruption of thiol balance within the cell, thus causing
cellular redox imbalance and ultimately oxidative stress. This leads to significant increases
in cell killing, causing the GNPs to act as radiosensitizers. Variation in the expression
levels of PDI in cancerous cells provides some insight into the range of radiosensitization
observed across cell types.
In the next section, we highlight situations where radiosensitization enhancement has
been achieved resorting to nanoparticles.
Gold nanoparticle-mediated enhanced radiosensitization has been achieved by several
It was found using 50-nm GNPs that radiosensitized HeLa cells when irradiated
with 220 kVp X-rays giving a DEF (dose enhancement factor) of 1.43 greater than that
observed for smaller nanoparticles (from 14 to 74 nm). The cellular uptake rate was
higher for the 50-nm GNPs, which was correlated with an increased
radiosensitization, compared to the smaller GNPs tested, and concentration dependent (Chithrani
et al. 2010). Also, functionalized Glu (thioglucose)-GNPs and AET (cysteamine)-GNPs
exposed to 200 kVp X-rays and gamma rays demonstrated a significant increase in cell
death in breast cancer cells compared to naked GNPs (Kong et al. 2008). Further
experiments demonstrated that 1.9-nm GNPs (AuroVistTM) following 2 Gy radiation exposure
(225 kVp) radiosensitize cells increasing apoptotic levels (Taggart et al. 2014). Moreover,
in vitro studies have demonstrated GNPs’ ability to radiosensitize tumours at clinically
relevant energies. At highest energies (6 MV), this has been shown by Chithrani et al.
(2010). This is supported by 1.9-nm GNPs (AuroVistTM) that radiosensitize at 6 MV,
and 15 MV X-rays with DEFs of 1.29 and 1.16 in MDA-MB-231 cells (Jain et al. 2011).
Hainfeld demonstrated evidence of radiosensitization in mammary tumour-bearing
mice using 1.9-nm GNPs and 250 kVp X-ray radiation. Mice irradiated together with
GNPs had 86% 1-year survival contrasting with 20% for X-rays alone (Hainfeld et al.
2004). Furthermore, citrate-coated GNPs that had a low radiosensitization effect in vitro
with a DEF of 1.08 promoted a delay in tumour growth in B16F10 murine melanoma
model. This was accompanied with increased survival from 20 days for non-irradiated
mice to 55 days for mice irradiated with 6 MV and 65 days for mice irradiated with
GNPs (Chang et al. 2008).
Although radiosensitization can be observed in some cell lines, others like DU145
human prostate cancer cells that uptake GNPs do not show significant effects at kV
nor at MV energies (sensitization enhancement ratio: 0.97–1.08) (Jain et al. 2011). A
summary of radiosensitizing experiments combining GNPs and ionizing radiation is
presented in Table 1. Cumulatively, these data strongly suggest a significant biological
component in GNP radiosensitization; however, the exact cellular mechanisms remain
to be fully elucidated.
Cell cycle effects
GNPs may enhance radiosensitization by causing cell cycle disruption and inducing
apoptosis. The sensitivity and consequent biological effects of radiation exposure are
dependent on the cell cycle phase. Different cell cycle phases present differential
radiation sensitivity with late S-phase cell being the most radioresistant and late G2 and
mitosis being the most sensitive (Pawlik and Keyomarsi 2004). In response to radiation, cells
activate cell cycle checkpoints in G1, S and G2 phases in order to repair genomic defects,
maintaining its integrity, or prevent cell division by activating cell death mechanisms
(Kastan and Bartek 2004). Materials other than gold, such as tachpyridine, have been
shown to induce cell cycle arrest in G2/M phase most likely due to its metal binding
activity (Turner et al. 2005). However, GNPs have been more extensively studied leading
to several other reports of cell cycle distribution alterations (Geng et al. 2011; Roa et al.
2009; Kang et al. 2010; Mackey et al. 2013; Mackey and El-Sayed 2014; Ganesh Kumar
et al. 2015).
So far, very few studies have been reported to analyse the effects of GNPs in the
cell cycle after radiation exposure. Roa et al. (2009) found that GNPs (Glucose-GNPs,
10.8 nm) alone can promote an increase in the G2/M phase in DU-145 cancer cells.
When irradiated using a Cs-137 source, G0/G1 phase has been shown to accelerate and
arrest DU-145 cells in the G2/M phase. In these cells, increased expression of cyclin
Table 1 Summary of radiosensitizing experimental data obtained with ionizing radiation
and gold nanoparticles
Surface coating Cell model
10 mg/ml, 15 min Thiol
Liu et al. (2015) 14.8
662 keV (137-Cs) 1.24–1.38
6 MV e−
Gamma (60-Co) 1.52
Carbon (62 MeV) 1.39
Surface coating Cell model
6 MeV e−
kinases (cyclin B1 and E) involved in the regulation of the cell cycle was found together
with a decreased expression of p53 (tumour protein 53) and cyclin A.
However, most studies have been performed in the absence of radiation and showed
distinct results (Wahab et al. 2014; Arnér and Holmgren 2000; Taggart et al. 2016; Kang
et al. 2010; Mackey et al. 2013; Mackey and El-Sayed 2014; Ganesh Kumar et al. 2015;
Cui et al. 2014). Thioglucose-coated 14-nm gold nanoparticles promoted an increase in
the G2/M cell phase, compared to the control, which lead to enhanced SK-OV-3 cell
sensitivity to 6 MV X-ray exposure (Geng et al. 2011). The effects of nuclear-targeted GNPs
have also been investigated. The results indicated that nanoparticles alone increase in the
sub-G1 population and disruption of the G1/S transition inducing apoptosis in cancer
cells (Kang et al. 2010; Mackey et al. 2013). Another study involving 30-nm NLS (nuclear
localization sequence)-GNPs in human oral squamous carcinoma (HSC-3) has shown
an increase in S phase and a decrease in G2/M phase sub-population. In combination
with 5′-fluorouracil that is active during the S phase, these cells were chemosensitized
(Mackey and El-Sayed 2014). Furthermore, bacteria-mediated anti-proliferative GNPs
demonstrated G2/M arrest accompanied with the inhibition of tubulin polymerization
and increased activation of caspases 8, 9 and 3, in DU145 cells, suggesting increased
apoptosis levels (Ganesh Kumar et al. 2015). Despite the results demonstrating GNPs’
influence in the cell cycle, there are other reports indicating no significant interference
(Pan et al. 2009; Cui et al. 2014; Butterworth et al. 2010). This has been reported with
2.7-nm tiopronin-GNPs and 1.4-nm triphenyl monosulfonate-GNPs (Pan et al. 2009;
Cui et al. 2014). However, the results might be cell line dependent as found using
1.9nm GNPs (AuroVistTM) and two different cell lines, DU-145 and MDA-MB-231 cells.
An increase in the sub-G1 population of DU-145 cells was seen after 48-h incubation,
whereas this was not detected in MDA-MB-231 cells (Butterworth et al. 2010). Again,
the coating and size of the nanoparticles induce distinct responses in the various cell
lines. The variety of concentrations, coatings, materials and cell lines makes it very hard
to draw any conclusions regarding the exact mechanism of action of NPs. However, the
alterations induced by GNPs in cell kinetics could be associated to the accumulation
in G2/M which is known to be the most radiosensitive, thus increasing GNP-mediated
DNA damage and repair
Another mechanism involved in GNP-induced radiosensitization is DNA damage and
repair. Radiation itself induces double-strand breaks (DSBs) in DNA and their repair is
essential for cell survival. Given the importance of DNA stability in determining cellular
propagation, it is a key target for agents that attempt to halt cancer cells from
dividing. Thus, cytotoxic targeting DNA agents such as cisplatin, gemcitabine and mitomycin
C have been tested regarding their ability to act as radiosensitizers (Choudhury et al.
2006). The induction of DSBs has also been reported in the presence of GNP γ-H2AX
foci analysis (Chithrani et al. 2010; Banáth and Olive 2003). Early DNA damage (1 h
post irradiation) caused by GNPs appears to be related to its presence in the
perinuclear region at the time of irradiation. However, late DNA damage (24 h post irradiation)
seems to be related to other indirect processes such as radical production after
interaction with water (Mcquaid et al. 2016).
Studies conducted with 50-nm citrate GNPs demonstrated increased foci number
after being irradiated with 220 kVp and 6 MV energies for 4 or 24 h. The increased
residual damage suggests possible inhibition or delay in DNA damage induction and/
or repair which can be essential to the radiosensitization mechanism of NPs (Chithrani
et al. 2010).
This has been further confirmed with 2.7-nm tiopronin-GNPs irradiated with 250 kVp
X-rays. An increased residual DNA damage after being irradiated for 24 h was found in
the samples where NPs were present. Contrastingly, no significant effect was observed
only at 30 min post irradiation suggesting that the GNPs had no influence on the
induction of DNA damage (Cui et al. 2014). Furthermore, GNPs have been found to induce
DSBs in hepatocellular carcinoma cells after radiation exposure. Residual damage has
also been found when the cells were irradiated in the presence of nanogold indicating
influence in the repair mechanisms of the cells (Zheng et al. 2013). GNPs might not have
all the same mechanisms of action and may induce distinct repair kinetics across
different cell lines. For instance, BSA (bovine serum albumin)-capped GNPs induce increased
γ-H2AX foci after 2 or 4 h of irradiation but no change is observed in the samples
incubated for 24 h. This implies that these NPs do not influence cellular repair mechanisms
(Chen et al. 2015). Other NPs such as 1.9-nm (AuroVistTM) have been reported to have
no impact on the formation or repair in DSBs, neither at 1 h nor at 24 h after irradiation,
in MDA-MB-231 cells (Jain et al. 2011).
Even if NPs demonstrate a radiosensitizing potential by promoting dose enhancement
and potentially contributing to increased DNA DSB formation, the lack of consistency of
cell lines, radiation sources and energies, treatment conditions and nanoparticles
properties lead to incomparable results, making it difficult to draw a conclusion. The
understanding of how different properties of the GNPs, irradiation conditions and biological
response of various cell lines contribute to DNA damage and repair could further shed
light on the underlying mechanism of GNP influence on DNA damage response.
Potential impacts of bystander effects of GNP radiosensitization
In addition to direct radiation effects, communication between cells is also very
important after radiation exposure. Cells that have not been directly exposed to radiation can
receive signals from irradiated ones that were in the vicinity responding in a similar way
to direct exposure (Najafi et al. 2014). This process is called the bystander effect and it
can occur in different cell types such as endothelial cells, fibroblasts, lymphocytes and
tumour cells (Havaki et al. 2015; Prise and O’Sullivan 2009; Butterworth et al. 2013). The
bystander signals involved in this process may cause altered gene expression, damage in
the DNA and chromosomes, cell proliferation alterations, cell death or changes in the
translation process in non-irradiated cells (Najafi et al. 2014).
The main bystander signalling molecules are reactive oxygen species or nitrogen
reactive species (RNS), cytokines, miRNA (micro-ribonucleic acid) or extracellular
oxidized DNA (ecDNA) (Azzam et al. 2002, 2004; Barber 2011). These are released into
the surrounding environment and reach the bystander cells through passive diffusion
or by binding to receptors on their plasma membrane (Barber 2011; Azzam et al. 2003).
Also it can occur by direct cell-to-cell contact via gap junction intercellular
communication (GJIC) (Azzam et al. 2001). Furthermore, exosomes carrying miRNA are believed
to mediate intercellular signalling between tumour cells and bystander cells (Melo et al.
2016; Yang et al. 2011; Umezu et al. 2013; Sánchez et al. 2015). 21 miRNAs have been
found recently to be up- or downregulated after ionizing radiation exposure.
Extracellular miR-1246 (micro-ribonucleic acid 1246) in particular appears to be increasing with
irradiation dose. It was found to enhance proliferation and resistance in lung cancer cells
by targeting death receptor 5 (DR5) although through a non-exosome associated
pathway (Yuan et al. 2014). Nevertheless, soluble miRNAs involved in bystander signalling
can be generated due to ROS, RNS, cytochrome c and cytokines (Hei 2015; Shao et al.
2011; He et al. 2011).
As NPs have been shown to alter cytokine and gene expression as well as ROS
production, their single presence in the tumour environment could further change the way cells
respond to radiation. NPs could also mediate bystander signalling, for example, small
titanium dioxide NPs induce higher levels of oxidative stress and production of
inflammatory cytokines. The expression of cytokine macrophage inflammatory protein-1 alpha
(MIP-1α) and high-mobility group protein 1 (HMGB1) were found to be expressed in
the presence of these NPs, both in vitro and in vivo (Fujiwara et al. 2015). This may play
a role in enhancing oxidative stress as HMGB1 is known to be an inflammatory
macrophage-secreted cytokine, which is activated by TNFα (tumour necrosis factor alpha)
and IL-1β (interleukin 1 beta) (Andersson et al. 2000; Tang et al. 2009). The latter will in
turn promote the secretion of HMGB1 (Tang et al. 2009). This creates a cycle that can
propagate inflammation and it might also propagate bystander effects as cytokines like
TNFα and IL-1β have been shown to be elevated in bystander cells (Zhou et al. 2008).
The increased production of IL (interleukin) and TNFα stimulates nitrogen oxide (NO)
and ROS biosynthesis by activating NF-kB (nuclear factor kappa B) transcription
factor, directly or indirectly, which in turn leads to the activation of iNOS (inducible nitric
oxide synthase) and COX-2 (cyclooxygenase-2) genes’ expression (Zhou et al. 2008).
iNOS controls the production of NO and COX-2 is involved in ROS production,
therefore increasing oxidative stress (Najafi et al. 2014; Hei et al. 2011). Moreover, small
airway epithelial cells (SAECs) exposed to GNPs are able to induce protein expression in
neighbouring lung fibroblasts in co-culture systems. This study found that 47 proteins
were upregulated, while 62 were downregulated in the fibroblasts receiving signals
from the incubated SAECs with GNPs. Most of the proteins identified are involved in
cell adhesion, extracellular matrix and cytoskeleton remodelling. Plasminogen
activator, PLAU (plasminogen activator urokinase), UPA (urokinase-type plasminogen
activator) and GRO-1 (growth-regulated oncogene 1) that are implicated in cell migration
were found to be downregulated, potentially decreasing it. Contrastingly, proteins that
promote cell adhesion such as Paxillin (PXN), breast cancer anti-oestrogen resistance 1
(BCAR1) and Caveolin-1 (Cav-1) were upregulated (Ng et al. 2015).
PXN, a focal adhesion (FA), is an associated adapter protein that regulates cell
spreading and motility. BCAR1 has been correlated with controlling the spread and motility
of cancer cells through regulating FA (Machiyama et al. 2014; Miao et al. 2012; Schaller
2001). Furthermore, Cav-1 is known to mediate cancer metastasis (Brennan et al. 2012).
Also Cav-1 can promote NF-kB activation and lung inflammatory response, through
eNOS (endothelial nitric oxide synthase) and NO production (Mirza et al. 2010). These
results might indicate airway inflammation since it can be related to increased adhesion
molecules (Garrean et al. 2006; Liu et al. 2014). The elevated cell adhesion was
accompanied by altered F-actin stress fibre arrangement in the cytoskeleton of the lung
fibroblasts. Similarly, there was an increase in vinculin binding sites that are associated with
F-actin anchoring to the cell membranes of the fibroblasts, which can potentially lead
to enhanced vascular permeability as previously described (Snyder-Talkington et al.
2013; Pacurari et al. 2012; Setyawati et al. 2013). Overall, cytoskeleton remodelling and
increased cell adhesion may affect lung function, thus reminding of the importance of
understanding the cellular communication pathways in the presence of NPs even in the
absence of radiation (Ng et al. 2015).
In addition to cellular bystander responses at the tissue and whole organism levels, a
phenomenon called an abscopal effect can occur. There is a response of an organ/site
distant from the irradiated one, where the cells are not close to each other, which has
been observed in patients undergoing localized radiotherapy (Kaminski et al. 2005).
The importance of the abscopal effect remains to be fully understood. It might have the
potential to either increase cell killing or protect normal tissues (Prise and O’Sullivan
2009). The role of NPs in mediating abscopal effects has not been elucidated due to a
lack of in vivo and clinical studies.
As nanoparticles may induce changes in cell signalling, leading to various responses
depending on their size, shape and coating, understanding which signalling pathways
are influenced could potentially help understand their mechanism of action in the
bystander/abscopal and radiosensitization effects.
Uptake, imaging potential and toxicity of GNPs in biological systems
Radiation absorption and dose deposition is thought to be partially reliant on the
number of gold nanoparticles present within the cell, meaning that cellular uptake and
distribution of GNPs will have a direct influence on the degree of radiosensitization observed
(Chithrani and Chan 2007). This makes cellular uptake of GNPs an important metric
in modifying sensitivity to radiotherapy. Modelling carried out to replicate GNP uptake
equal to 1% mass in the cytosol demonstrated dose enhancement in both the nucleus
and mitochondria, despite cytosol localization. It was concluded that the physical
mechanism of dose enhancement was caused by photoelectron delocalization from the
cytosol to cell organelles, meaning that the dose enhancement effects were not limited to the
vicinity of the nanoparticles (McNamara et al. 2016).
Both uptake potential and blood circulation times have been shown to be closely
associated with nanoparticle size. The optimal size for uptake has been found to be between
25 and 50 nm, with particles smaller than 10 nm or larger than 100 nm exhibiting a
reduced uptake potential (Yang et al. 2014).
Chithrani et al. found the uptake of gold nanoparticles to be heavily reliant on their
size and shape. Internalization of smaller particles is observed to be a more rapid process
than that of larger particles. This is important as the uptake of nanoparticles into the cell
will have a direct influence on the level of radiosensitization (Chithrani and Chan 2007).
Coulter et al. reported the maximum amount of nanoparticle uptake to occur within
the first few hours of exposure of cells to 1.9-nm GNPs, with a plateau being reached
after 6 h. However, they also highlighted the difficulty in making direct comparisons
with previous work as a result of the number of variables involved, as uptake may differ
depending on the nanoparticle shape, size, coating, concentration and charge, as well
as cell type (Coulter et al. 2012). These studies show cellular uptake to be dependent on
concentration, time and cell type. Experiments carried out in hypoxic cell models saw
reduced GNP uptake, thought to be linked to reduced energy production in the cells
(Jain et al. 2014).
While GNPs will passively accumulate in tumours due to the EPR effect, their
encapsulation within liposomes has been shown to result in higher cellular internalization
(Maeda et al. 2003). Uncapped nanoparticles bind to various plasma proteins upon
administration, resulting in a large number being internalized by macrophages and
removed from circulation. Liposomes have a history of being used for drug
encapsulation and delivery as their large size (100–200 nm) ensures that they can pack many
GNPs within their lipid bilayers. Small nanoparticles encapsulated within liposomes
show better passive accumulation within the tumour than non-encapsulated
nanoparticles (Chithrani et al. 2010). The contents can then be released through a triggering
technique, allowing the nanoparticles to penetrate the tumour tissue more effectively (Kneidl
et al. 2014). Alternatively, nanoparticles capped with PEG (polyethylene glycol) alone
will show an increase in their half-life in blood (Hirn et al. 2011), while HSA (human
serum albumin)-conjugated gold nanoparticles have shown increased retention in the
lungs and brain compared with both apoE (apolipoprotein E)-capped and
citrate-stabilized nanoparticles (Schuffler et al. 2014).
In contrast to passive targeting, active targeting is the functionalization of the GNP
surface with peptides, ligands or antibodies in order to preferentially target tumour
cells (Schuemann et al. 2016), taking advantage of the overexpressed surface receptors
of cancer cells. Not only does this increase the therapeutic ratio through achieving a
greater nanoparticle concentration within the tumour, but also reduces the overall
volume of gold required for treatment. Sykes et al. (2014) carried out an investigation into
the impact that nanoparticle size has on active and passive targeting. Gold
nanoparticles with diameters of 15, 30, 60 and 100 nm were prepared with the surface modified
with either PEG or PEG conjugated to transferrin. Those nanoparticles modified with
transferrin showed a significant increase in tumour accumulation in vivo, with
accumulation in the 60-nm particles being 1.9 times greater than that in their passive
counterparts. However, it is of note that while the PEG-only nanoparticles were slower, they also
diffused deeper into the tumour. Popovtzer et al. (2016) successfully demonstrated
significant improvement in tumour radiosensitivity using GNPs covalently conjugated to
CTX monoclonal antibody in mice. This actively targeted the tumour with no evidence
of early or delayed toxicity, confirmed by histological characterization of the tumour and
adjacent tissue both 1 and 6 weeks post treatment. TNF has been covalently conjugated
to gold nanoparticles, with the aim of the interaction between TNF and its receptor
TNF-R1 causing active targeting of the tumour cells. Molecules of PEG-Thiol are
interspersed between TNF molecules, and nanoparticles may be treated with a coating to
increase cellular internalization.
Tumour blood vessels may also be promising targets, as sub-100-nm-diameter
nanoparticles are expected to accumulate in the vasculature (Perrault et al. 2009). The
important role of endothelial cells within the tumour vasculature makes them ideal targets
with high potential clinical impact. However, a model generated by Berbeco et al.
examining gold nanoparticles as tumour vascular disrupting agents found the boosted
irradiation of endothelial cells alone was not viable due to the short range of the ionizing
particles. The combination of tumour-targeting functionalization with image-guided
radiotherapy could combat this by ensuring that the tumour has maximum levels of
GNPs present during irradiation. Nanoparticle-mediated drug delivery to the tumour
vasculature has been shown to have anti-metastatic effects and tumour gold content will
give a good indication of overall tumour vascularity (Murphy et al. 2008). Shifting
nanoparticle accumulation from the reticuloendothelial system (RES) to other organs will
result in longer retention times, though unanticipated retention over prolonged periods
of time may result in cytotoxic effects (Balasubramanian et al. 2010). This accumulation
of GNPs may be achieved through the addition of peptides, allowing more site-specific
delivery. For example, insulin has been used to improve the delivery of gold
nanoparticles to target sites in the brain (Shilo et al. 2014), while other peptide-capped gold
nanoparticles have been shown to pass through the blood–brain barrier using a number of
mechanisms (Velasco-Aguirre et al. 2015). The ability to accurately target organ sites for
uptake greatly enhances the radiosensitizing potential of GNPs.
In conclusion, while there are several benefits to passive targeting, it is noticeably less
efficient in slow-growing models when compared to fast-growing models, since the
former have more mature and intact blood vessels (Kunjachan et al. 2015). Cellular
targeting can face problems in the way of tissue barriers which vascular targeting avoids
by providing nanoparticles with direct access or binding to the overexpressed targets.
Attacking the vasculature can also have the added benefit of affecting the numerous
cancer cells that rely on it for growth (Kunjachan et al. 2014, 2015). GNP uptake has
been shown to be reliant on several variables, including nanoparticle shape, size, charge
and concentration, alongside the cell type. While the EPR effect results in the passive
accumulation of GNPs within the tumour, encapsulation and specific coatings allow for
increased cellular targeting and retention.
A consequence of the increasing use of gold nanoparticles in biomedical applications is
the need for their accurate and efficient detection in both biological and tumour
samples. The ability to determine nanoparticle location provides insights of uptake pathways
as well as potentially identifying nanoparticle location as a cause of cytotoxicity.
Accurate imaging of nanoparticle location within a sample will help achieve precise dose
deposition and may also provide understanding of mechanisms behind radiosensitization.
To quantify the uptake and distribution of nanoparticles within cells, several imaging
techniques can be used. The physical properties of gold nanoparticles also allow them to
be used as imaging agents. Miladi et al. (2014) used 2.4-nm GNPs coated with DTDTPA
(dithiolated diethylenetriamine pentaacetic acid)-gadolinium chelates to combine MRI
(magnetic resonance imaging) and radiosensitizing effects. The DTDTPA coating acts
in preventing the clumping of GNPs along with slowing their uptake by the RES.
Osteosarcoma- and gliosarcoma-bearing rats were injected with the nanoparticles, which were
then monitored using MRI. The rats were then irradiated at the point when the highest
content of nanoparticles was observed in the tumour. Several approaches can be used
to image the uptake and distribution of GNPs throughout the cell, including localized
surface plasmon resonance, photoacoustic imaging, computerized tomography, X-ray
fluorescence computed tomography and electron microscopy. Each technique will have
individual advantages and limitations, so it can be worthwhile to apply multiple
techniques in order to improve the reliability of diagnostics and treatments (Botchway et al.
A characteristic affecting the imaging techniques that can be used with gold
nanoparticles is localized surface plasmon resonance (LSPR). This is instigated by their ability to
absorb and scatter specific wavelengths of light and refers to the resonance established
between incident light photons and particle surface electrons. The LSPR of a
nanoparticle can provide information about its overall size and structure, as when these change,
so does its resonant frequency. While LSPR has a high sensitivity and relatively low
cost, it can be time consuming to set up depending on the target (Petryayeva and Krull
2011). Photoacoustic imaging (PA) involves irradiating tissue using a nonionizing
shortpulsed laser beam. Exogenous contrast agents absorb this energy to produce ultrasound
waves, which are received using a transducer. The mechanical acoustic waves are then
converted into an electronic signal which is processed to form an image. As the
photoacoustic waves are only generated within the tissue sample, there is reduced background
interference. PA gives higher spatial resolution and deeper imaging depth compared to
fluorescence optical imaging, while the lack of ionizing radiation also makes it a safer
option than computerized tomography (CT) (Wang 2008; Pan et al. 2013; Li and Chen
The strong effect demonstrated by gold nanoparticles allows them to be used as
contrast agents in photoacoustic imaging. Changing the shape and size of the nanoparticles
that are being used enables the tuning of the magnitude of light being absorbed and
scattered and when compared with other contrast agents, such as imaging dyes and silver
nanoparticles, they demonstrate a greater absorbance (Menon et al. 2013; Huang and
The accumulation of gold nanoparticles in tumours due to the EPR makes them well
suited for photoacoustic imaging. This helps determine the location of the tumour along
with assessing the vasculature and accumulation of therapeutic agents. However, the
photostability of gold nanoparticles is a potential limitation as they can change shape
due to high laser energies. Zhang et al. utilized GNPs as PA agents to detect human
breast cancer xenografts in mice. The nanoparticles were found to accumulate within
the tumours after 5 h following injection via tail vein and a significant enhancement in
signal intensity was seen. This accumulation and signal enhancement would allow gold
nanoparticles to be used as both tumour contrast agents and mediators of cancer
therapy (Chang et al. 1999; Zhang et al. 2009).
The high atomic number and potentially long circulation time make gold
nanoparticles ideal contrast agents for CT (Cormode et al. 2014). Hainfeld et al. used 1.9-nm gold
nanoparticles as a contrast agent for X-ray therapy in rats. Contrast was estimated to be
~3 times greater than that of iodine at 100 keV, a useful range for clinical CT. The agent
was then observed to be excreted via the kidneys with no observed toxicity. Further
work used gold nanoparticles as a contrast agent to detect smaller tumours (1.5 mm).
Nanoparticles were observed to accumulate in the tumours, and micro-CT allowed for
quantification of this. They also noted that there was a greater uptake of nanoparticles
without any attached antibody, which they believed to be due to the smaller size.
Alric et al. used a gadolinium chelate to produce a nanoparticle that could be used for
both MRI and CT imaging. The nanoparticles were found to circulate freely in blood
with no adverse accumulation in the lungs, liver and spleen (Alric et al. 2008).
X-ray fluorescence computed tomography (XFCT) is an imaging technique that aims
to simultaneously determine the identity, quantity and spatial distribution of elements
within imaged objects. Previously, Cheong et al. had been successful in accurately
identifying the location of a GNP-filled object within a small animal-sized plastic phantom,
as well as quantifying the amount of GNPs present. However, at the time their technique
was not yet practical for routine in vivo use (Cheong et al. 2010). More recently,
Manohar et al. (2016) demonstrated the use of benchtop XFCT for imaging a small animal
that had been injected with gold nanoparticles. However, they found that their set-up
required further refinement before it could be used routinely.
Electron microscopy is well suited to imaging gold nanoparticles due to their high
electron density. It allows for the determination of the size and shape down to 1 nm and
as a result is commonly used when characterizing nanoparticles. However, transmission
electron microscopy (TEM) has a complicated and time-consuming sample preparation,
and individual nanoparticles are not also distinguishable if their size is below the
resolution limit (Schrand et al. 2010). Scanning electron microscopy (SEM) requires coating
samples in a conductive metal, usually composed of nanometer-sized clusters. The
similarities between samples prepared using conventional methods and metal nanoparticles
make SEM incompatible. However, Goldstein et al. (2014) developed a simplified
procedure that involved chromium coating, allowing them to observe cellular uptake of GNPs.
The physical properties of GNPs allow them to be used with a wide range of imaging
techniques. This not only allows for the quantification of their uptake and distribution,
but also makes them potential contrast agents. The advantages and limitations of each
technique indicate that it may be necessary for more than one technique to be applied to
GNPs must exhibit safe behaviour after cellular uptake to be considered potential
radiosensitizers. If individual nanoparticles are found to be cytotoxic or to reduce cell
viability, then they might not be suitable for clinical use. Following IV (intravenous)
injection, both the biodistribution and clearance of nanoparticles are influenced by
various physiological factors (bloodstream, opsonization, endothelial permeability of vessels
and organs) as well as the individual physicochemical properties of nanoparticles (size,
charge and surface chemistry). These physicochemical properties can be altered to
control their biodistribution. It is preferable that particles are later removed through urine
as this implies that there has been no degradation.
There is a level of uncertainty regarding the cytotoxicity of GNPs. While bulk gold
is known to be biologically safe, functionalized GNPs have shown obvious cytotoxicity
(Goodman et al. 2004). Size, concentration, cell type and treatment time are all basic
parameters to be considered when examining the cytotoxicity of GNPs. Size is an
important factor, as very small particles have been found to be highly toxic, and larger
particles are relatively nontoxic (Pan et al. 2007).
Zhang et al. summarized that while high gold concentrations cause an obvious
decrease in cell viability, low gold concentrations do not appear to influence viability.
Nanoparticles with a diameter of 15 nm were found to be nontoxic up to 75 µg/ml,
though cell viability was obviously affected at concentrations of gold >150 µg/ml. At
600 µg/ml, cell viability was reduced to 41.8%, compared to 93.9% at 18.75 µg/ml (Zhang
et al. 2009).
A study by Connor et al. found no difference in growth rates between untreated cells
and cells exposed to 25 mM concentration of 18-nm nanoparticles over the course of
5 days. The uptake and localization of the nanoparticles in the cell were confirmed by
TEM, leading to the conclusion that nanoparticles are not inherently toxic to human
cells. However, it was noted that the determination of whether the nanoparticles are
modified by their environment is important, as this may result in significant variation to
their clinical applications (Connor et al. 2005).
In 2004, Hainfeld et al. (2004) carried out work that involved injecting mice with
2.7 g Au/kg per body mass. These mice survived over a year without showing any
obvious clinical effects, suggesting that in this case the nanoparticles were biocompatible.
Further work by Hainfeld et al. (2013) showed that irradiation with 11.2-nm
nanoparticles increased long-term survival (over 1 year) of mice by 50%, while none of the mice
receiving the same treatment without nanoparticles survived longer than 150 days. As
the LD50 (lethal dose at 50%) of these nanoparticles was >5 g Au/kg, a dose of 4 g Au/
kg was used, showing that by using the appropriate dose of nanoparticles, toxicity can be
averted and lifespan can be greatly improved.
The effects of 5- and 15-nm GNPs on mouse fibroblast cells were examined by
Coradeghini et al. to provide data on the toxic potential of different sized nanoparticles. Using
a colony forming efficiency (CFE) assay, the in vitro toxicity of gold nanoparticles was
tested at the concentrations of 10–300 µM and at the times of 2, 24 and 72 h. Significant
cytotoxicity was only seen in cells treated with 5-nm nanoparticles at a concentration
over 50 µM and an exposure time of 72 h, with no significant cytotoxicity observed in
the 15-nm nanoparticles. TEM imaging showed that cellular internalization occurred
for both sizes of nanoparticles (Coradeghini et al. 2013).
Stefan et al. examined the effects of 12- and 22-nm chitosan-capped gold
nanoparticles on rats treated with lipopolysaccharide (LPS). Brain and liver tissue reactivity was
assessed following 8 days of administration. They found that while the body weight of
the treated rats did not change significantly, the ratio between liver and body weights
significantly increased with both nanoparticle sizes, especially the 22-nm ones,
suggesting potential liver toxicity. The 22-nm nanoparticles also experienced a significant
decrease in their brain-to-body weight ratio, suggesting possible brain damage.
Darkfield imaging showed the agglomeration of nanoparticles within cytoplasmic cellular
regions as a potential cause of this damage. This was not seen when using the 12-nm
nanoparticles (Stefan et al. 2013).
While it remains a challenge to accurately estimate the cellular response to a given
nanoparticle size, there are general trends which can be trusted. While both the cell type
and surface properties of the nanoparticle play a role in nanoparticle uptake, smaller
nanoparticles are more likely to be passively internalized, though are also more likely to
have a cytotoxic effect (Shang et al. 2014).
As there is increasing interest in using nanomaterials in both research and a clinical
setting it is important that nanoparticle cytotoxicity can be tested in a fast and efficient
manner. Karlsson et al. used the in vivo assay “ToxTracker” with a panel of metal oxide
and silver based nanoparticles. This comprises of a panel of mouse embryonic stem cells,
each containing a GFP (green fluorescent protein)-tagged reporter for a distinct cellular
signalling pathway. In this way it is possible to identify DNA damage caused by direct
DNA interaction, oxidative stress and general cellular stress (Karlsson et al. 2014). If this
assay was adapted for GNPs it may elucidate their cytotoxic properties.
Despite NPs’ potential to induce radiosensitization in cancer cells, there are several
challenges towards clinical translation which has, to date, led to only a few clinical trials
being undertaken with the majority being liposome based and related to targeting, not
involving radiosensitization (Anselmo and Mitragotri 2016). Among these challenges,
there are some inconsistencies found in the mechanisms of action of different GNPs,
reduced long term side effects in vivo studies and the limited demonstration of
therapeutic efficacy at megavoltage energies, at which radiotherapy is clinically performed
(McMahon et al. 2016).
Regardless of the distinct simulation results using megavoltage energies, an
experimentally significant increase in DNA damage in the presence of GNPs has been
observed. At 6 MV, the number of DSBs increases in HeLa cells, with increasing depth
and as a function of the field size (Berbeco et al. 2012). Also, at MV energies it is possible
to achieve dose enhancement in vivo (Chang et al. 2008; Mousavie Anijdan et al. 2013).
Furthermore, a number of studies conducted at MV energies are also required prior to
clinical application to support the results obtained at keV energies that most
experiments are performed at. Also the dose delivered needs to be adjusted as NPs increase
the dose deposition tremendously in its vicinity and in a clinical context it is usually
much lower to limit exposure of organs at risk (McMahon et al. 2011). Moreover, NPs
may change cellular communication influencing the clinical outcome, therefore
requiring further studies in this field.
Nevertheless, using NPs can be an asset not just to radiosensitize cells but also to
provide contrast as they can be imaged. This would provide a theranostic agent,
combining therapeutic and imaging potential in one NP, thus improving accuracy and results of
treatment delivery. Despite its potential applications, without understanding the
mechanisms mediating the biological effects in cells, it is difficult to move towards clinical
applications in a robust way.
AET: cysteamine; apoE: apolipoprotein E; BCAR1: breast cancer anti-oestrogen resistance 1; BSA: bovine serum albumin;
Cav-1: caveolin-1; CT: computerized tomography; COX-2: cyclooxygenase-2; DSBs: double-strand breaks; DEF: dose
enhancement factor; DNA: deoxyribonucleic acid; DR5: death receptor 5; DTDTPA: dithiolated diethylenetriamine
pentaacetic acid; ecDNA: extracellular deoxyribonucleic acid; eNOS: endothelial nitric oxide synthase; FA: focal adhesion;
GRO-1: growth-regulated oncogene 1; GJIC: gap junction intercellular communication; Glu: thioglucose; GNPs: gold
nanoparticles; GFP: green fluorescent protein; GSH: glutathione; HER2: human epidermal growth factor; HSS: human serum
albumin; HMGB1: high-mobility group protein 1; IL: interleukin; IV: intravenous; IL-1β: interleukin 1 beta; IMRT:
intensitymodulated radiotherapy; iNOS: inducible nitric oxide synthase; LD50: lethal dose at 50%; LPS: lipopolysaccharide; LSPR:
localized surface plasmon resonance; MRI: magnetic resonance imaging; miRNA: micro-ribonucleic acid; miR-1246:
micro-ribonucleic acid 1246; MIP-1α: macrophage inflammatory protein-1 alpha; NF-kB: nuclear factor kappa B; NO:
nitrogen oxide; NPs: nanoparticles; PA: photoacoustic imaging; PDI: protein disulfide isomerase; PEG: polyethylene
glycol; PLAU: plasminogen activator urokinase; PNX: paxillin; p53: tumour protein 53; RES: reticuloendothelial system;
ROS: reactive oxygen species; RNS: reactive nitrogen species; SAECs: small airway epithelial cells; SEM: scanning electron
microscopy; TEM: transmission electron microscopy; TrxR1: thioredoxin reductase 1; TNFα: tumour necrosis factor alpha;
UPA: urokinase-type plasminogen activator; XFCT: x-ray fluorescence computed tomography.
SR drafted the manuscript and drew the figures. CC drafted the manuscript and aided in the preparation of figures and
table. GS, KB and KMP helped to draft the manuscript. KMP outlined the manuscript and was responsible for design and
coordination. All authors read and approved the final manuscript.
The authors are grateful to the European Commission Framework 7 Programme (grant number EC FP7 MC - ITN - 608163
– ARGENT) for funding this work. CC is funded by a joint Studentship between the National Physical Laboratory and
Queen’s University Belfast (NPL422559).
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