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ORIGINAL RESEARCH ARTICLE
Inhaled Nanoparticles Accumulate At Sites Of
Vascular Disease
Mark R. Miller1†* and Jennifer B. Raftis,† Jeremy P. Langrish,1 Steven G. McLean,1
Pawitrabhorn Samutrtai,3 Shea P. Connell,1 Simon Wilson,1 Alex T. Vesey, 1 Paul H.B.
Fokkens,4 A. John F. Boere,4 Petra Krystek,5 Colin J. Campbell,3 Patrick W.F. Hadoke,1
Ken Donaldson,2 Flemming R. Cassee,4,6 David E. Newby,1 Rodger Duffin2# and Nicholas
L. Mills1#
1 BHF Centre for Cardiovascular Science, University of Edinburgh, Edinburgh, United Kingdom
2 MRC Centre for Inflammation Research, University of Edinburgh, Edinburgh, United Kingdom
3 EaStCHEM School of Chemistry, University of Edinburgh, Edinburgh, United Kingdom
4 National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands
5 Institute for Environmental Studies (IVM), VU University, Amsterdam, The Netherlands
6 Institute for Risk Assessment Sciences, Utrecht University, Utrecht, The Netherlands
† Joint and equal contribution to first authorship
# Joint and equal contribution to senior authorship
*Corresponding Author: Dr Mark R Miller, [email protected]
KEYWORDS: nanoparticle; translocation; gold; air pollution; cardiovascular;
atherosclerosis.
1
ABSTRACT
The development of engineered nanomaterials is growing exponentially, despite concerns
over their potential similarities to environmental nanoparticles that are associated with significant
cardiorespiratory morbidity and mortality. The mechanisms through which inhalation of
nanoparticles could trigger acute cardiovascular events are emerging, but a fundamental
unanswered question remains; do inhaled nanoparticles translocate from the lung in man and
directly contribute to the pathogenesis of cardiovascular disease? In complementary clinical and
experimental studies, we used gold nanoparticles to evaluate particle translocation, permitting
detection by high-resolution inductively-coupled mass spectrometry and Raman microscopy.
Healthy volunteers were exposed to nanoparticles by acute inhalation, followed by repeated
sampling of blood and urine. Gold was detected in the blood and urine within 15 min – 24 h after
exposure, and was still present 3 months after exposure. Levels were greater following inhalation
of 5 nm (primary diameter) particles compared to 30 nm particles. Studies in mice demonstrated
the accumulation in the blood and liver following pulmonary exposure to a broader size range of
gold nanoparticles (2-200 nm primary diameter), with translocation markedly greater for particles
<10 nm diameter. Gold nanoparticles preferentially accumulated in inflammation-rich vascular
lesions of fat-fed apolipoproteinE-deficient mice. Furthermore, following inhalation, gold particles
could be detected in surgical specimens of carotid artery disease from patients at risk of stroke.
Translocation of inhaled nanoparticles into the systemic circulation and accumulation at sites of
vascular inflammation provides a direct mechanism that can explain the link between
environmental nanoparticles and cardiovascular disease, and has major implications for risk
management in the use of engineered nanomaterials.
2
The manufacture of nanomaterials is growing exponentially and with this so is the potential for
human exposure. The fate of engineered nanoparticles and effect on health following exposure are
largely unknown.1,2 Since manufactured nanoparticles of different types vary in their ability to cause
inflammation and toxicity in animal studies3 we may anticipate heterogeneity in their impact on
humans at plausible exposure. Given the prominent cardiovascular effects of nanosized particulate
matter in air pollution, our limited understanding of the potential cardiovascular effects of
engineered nanoparticles, in particular, is a pressing concern.4
Air pollution is a major public and environmental health issue contributing to up to 7 million
premature deaths worldwide each year.5,6 The adverse effects of particulate air pollution on
cardiovascular health have been established in a series of major epidemiological studies,7,8 and
even transient exposure to high levels of air pollution may trigger an acute cardiovascular event.9
Nanosized particulate matter in air pollution, such as that derived from vehicle exhaust, remains a
major public health concern.10,11 We have previously demonstrated that acute exposure to diesel
exhaust causes vascular dysfunction, thrombosis and myocardial ischemia in healthy individuals
and in patients with coronary heart disease.12,13 Chronic exposure to particulate air pollution is
associated with the development and progression of the vascular disease atherosclerosis in
animals and man.14,15 Whilst these observations are important, a major area of uncertainty remains.
How do inhaled particles influence the pathogenesis of systemic cardiovascular disease? Are
inhaled nanoparticles able to translocate into the circulation and directly contribute to
cardiovascular disease?
Inhaled nanoparticles are able to deposit deep in the lungs where they induce oxidative
stress and inflammation.16,17 It has been hypothesized that inflammatory mediators pass into the
systemic circulation18 and indirectly influence the cardiovascular system. However, evidence of
systemic inflammation following exposure is inconsistent and rather modest. An alternative
hypothesis is that inhaled nanoparticles penetrate the alveolar epithelium and translocate into the
circulation, and directly contribute to cardiovascular disease.19,20 It has been speculated that these
processes may be accelerated by inflammation, through increased permeability of the alveolar wall
3
or through assisted translocation within macrophages.21-23. Evaluation of nanoparticle translocation
following inhalation exposure is challenging because of the small size of particles, the low
deposited mass, and the requirement for extremely sensitive and specific methods of detection.
Previous studies in man have been inconsistent due to leaching of radiolabel from
nanoparticles.24,25 Whilst there is no evidence to date that translocation occurs in man, animal
studies have provided more compelling evidence that nanoparticles are able to translocate.20,26-27
Oberdorster and Kreyling report translocation and accumulation of particles in the liver.28,29
Whether this occurs in man and whether those particles entering the circulation interact with the
cardiovascular system is unknown, especially in relation to areas of the vasculature that are
susceptible to disease where their biological activity is of greatest risk.
Here we use complementary clinical and experimental studies to address whether inhaled
nanoparticles translocate from the lung into the circulation. Gold nanoparticles are used as a model
nanoparticles that are, available in relevant size ranges, effectively inert for controlled human
exposure, and to facilitate detection of low levels in systemic tissues through specific high
sensitivity techniques. We hypothesize that inhaled nanoparticles will gain access to the
bloodstream and accumulate at sites of vascular inflammation.
4
RESULTS & DISCUSSION
Particle translocation in man
Fourteen healthy male volunteers were exposed to gold nanoparticles (median primary
particle size of 3.8 nm, present in loose agglomerates of 18.7 nm (geometric standard deviation:
1.5; Figure 1A & 1B) by inhalation (116±12 µg/m3; 5.8±0.3 x 106 particles/cm3) for 2 hours during
intermittent exercise (Supplementary Figure S1). Gold exposure was well tolerated and there
were no adverse effects on lung performance (data not shown).
Blood and urine samples were obtained throughout the first 24 hours and at 3 months. No
volunteer had gold detectable in the bloodstream prior to exposure, but gold was detectable as
early as 15 min after exposure in some subjects, and was present in the majority (12/14 subjects,
86%) at 24 hours (Figure 1C). There was no evidence of a significant time-dependent increase in
gold concentration in the blood within the first 6 hours of exposure (Figure 1D). Gold was still
detectable in blood and urine at 3 months.
Particle translocation is dependent on particle size
To explore if the particle size affected their ability to translocate, we exposed healthy
volunteers to gold particles with a primary particle size of either ~4 nm (4.1±0.6 nm primary
diameter) or 34 nm (34±3 nm primary diameter) (n=10 and 9 volunteers, respectively; Figure 2A &
2B). Particles agglomerated in the aerosol to a median aerodynamic diameter of 18 nm and 52 nm,
respectively (Figure 2B). Gold was detectable in the blood at low levels following inhalation of
either size of particle, although levels of the smaller particle were far greater (Figure 2C). Gold was
detectable in the urine following exposure to 4 nm particles, but not in the urine of volunteers
exposed to the larger particulate (Figure 2D).
To address tissue accumulation over a wider range of particle sizes, we repeatedly instilled
mice with 2, 5, 10, 30 or 200 nm gold nanoparticles (primary particle size). Gold was detectable in
the blood following exposure to each size of particulate. However, the incidence of detectable gold,
5
and the concentration of gold, in the blood was far greater following exposure to the smaller
particles (Figure 2E). A threshold of particle accumulation (based on the limit of detection using
HR-ICPMS) was observed for a primary diameter of 10-30 nm. Particle accumulation in the liver
also followed a similar trend (Figure 2F), but, gold was only detectable in the urine following
exposure to particles ≤5 nm (Figure 2G).
Accumulation of translocated particles at sites of vascular inflammation
Given the close association of nanoparticles with phagocytic inflammatory cells, we wished
to explore whether inhaled nanoparticles accumulate at sites of vascular inflammation.
Apolipoprotein E knockout mice (ApoE-/-) were used as a well-established model of atherosclerosis,
fed a high-fat diet to accelerate the development of vascular lesions. Intra-tracheal administration
of gold nanoparticles (diameter 5 nm; BBInternational, UK; Figure 3A) or vehicle was conducted
twice weekly for 5 weeks to deliver a total of 110 µg of gold nanoparticles. Blood and liver samples
were taken 24 hours after the last instillation, and the aortic arch and descending aorta were
harvested as areas exhibiting and free from atherosclerosis, respectively.
Instillation of gold nanoparticles was associated with a mild pulmonary inflammatory
response (Figure 3B). At autopsy, gold nanoparticles were present in the alveoli and largely
contained within macrophages (Figure 4A & 4B). Gold was detected in the blood of gold-instilled
mice at concentrations of 9.4±1.2 ng/mL, compared with 0.4±0.2 ng/mL in vehicle-instilled mice
(P<0.0001; Figure 3C). Gold also accumulated in the liver (Figure 3C).
The aortic arch from ApoE-/- mice developed complex atherosclerotic plaques rich in lipids
and macrophage-derived foam cells. Gold concentrations in the aortic arch were 10-fold higher in
gold-treated compared to vehicle-treated mice (60±24 vs 6±1 ng/gram of tissue; P=0.023), and
exceeded concentrations in the descending thoracic aorta (12±6 ng/gram of tissue; Figure 3D).
Isolated histological sections of atherosclerotic plaque from gold-treated animals revealed the
presence of clusters of red-purple granules within foam cells (Figure 4C) similar in appearance to
nanoparticles in suspension. Particulate clusters were seen on transmission electron micrographs
of atherosclerotic plaque from gold-treated mice that were distinct from commonly demonstrated
6
electron-dense membranous structures (Figure 4D & Supplementary Figure S2). The presence
of particulate gold within atherosclerotic plaques was confirmed on silver-augmented arterial
sections of gold-treated, but not vehicle-treated, mice using Raman spectroscopy (Figure 5 &
Supplementary Figure S3).
In patients with a recent cerebrovascular accident scheduled to undergo carotid
endarterectomy, we evaluated particle translocation and accumulation at sites of atherosclerosis in
man (Figure 6A). Twelve patients were recruited, of which three were exposed to gold
nanoparticles (5 nm) for 4 hours under resting conditions 24 hours prior to undergoing surgery.
Gold was measureable at concentrations above the limit of quantification in blood from a single
patient and urine from 2 patients following gold exposure (Supplementary Figure S4). Raman
microscopy identified the presence of gold in the excised carotid plaque of those patients exposed
to gold nanoparticles, but not in those without prior exposure (Figure 6B & 6C).
Here we demonstrate that inhaled nanoparticles translocate from the lung into the circulation
in man, where they accumulate at sites of vascular inflammation. Particle translocation was size
dependent with greater translocation and accumulation of smaller nanoparticles. These
observations suggest a direct role of particle size for inhaled nanoparticles in the pathogenesis of
cardiovascular disease, and provide a mechanism whereby exposure to combustion-derived
nanoparticles and manufactured nanoparticles may promote atherosclerosis and trigger acute
cardiovascular events.
Evaluation of nanoparticle translocation following inhalation exposure is challenging.
Nemmar and colleagues previously demonstrated the presence of 99m technetium (99mTc) in the
bloodstream of healthy volunteers as early as one minute after inhalation of 99mTc–labeled carbon
nanoparticles.24 However, subsequent studies, could not replicate this finding30,25 suggesting that
soluble pertechnate salts, rather than nanoparticle translocation, accounted for the early and rapid
detection of radioactivity in the blood. We used insoluble gold nanoparticles as they are a similar
size to combustion-derived nanoparticles, have low biological activity, and have been used safely
in man as a radio-contrast agent and for drug-delivery.31 Endogenous levels of gold in biological
7
samples are negligible,32 permitting reliable identification and quantification of translocated gold at
nanogram levels using high-resolution inductively-coupled plasma mass spectroscopy and Raman
microscopy.
Our findings suggest that translocation of nanoparticles into the circulation occurs rapidly,
consistent with translocation via a passive transport mechanism, with a slow incremental
accumulation in systemic tissues. There was no evidence of a time-dependent increase in gold
concentration in the blood suggesting that whilst gold nanoparticles translocate into the circulation,
this is balanced by clearance. Indeed, gold was detected in the urine of almost all subjects within
24 hours at a mean concentration of 35±24 ng/mL. Based on the exposure characteristics of our
first study (2-h exposure of ~116 μg/m3 under light exercise), we calculate the total inhaled dose of
particulate to be ~690 mg. If ~80 µg of gold was cleared by the kidneys over 24 hours (average
volume of urine collected: 2.4 L containing 35 ng/mL), and, therefore, if this was the sole route of
clearance, we estimate that at least 0.2% of inhaled gold nanoparticles translocated from the lung
into circulation. This estimate is consistent with those derived from pulmonary instillation of
particles in animal models,33,34 and those derived from in vitro-in silico modeling.35 The majority of
inhaled nanoparticles are retained in the alveolar region and are slowly cleared over time.25 In the
present study, gold was still detectable in blood and urine at 3 months, consistent with ongoing
translocation of gold either through the alveolar membrane or possibly via the gastrointestinal tract
after clearance by the mucociliary escalator. These findings are consistent with those of Kreyling
and colleagues36,37 who demonstrated the persistence of radiolabelled iridium nanoparticles in the
lung 6 months after a 1-h inhalation in rats, with detectable levels in extra-pulmonary organs at this
time.
The physicochemical properties of the particle that permit translocation will have important
implications both for environmental particulates and manufactured nanomaterials. Particle size is
one of the most crucial parameters affecting the biokinetics and subsequent actions of
nanoparticles.38,39 Here we demonstrate in man, that there is less translocation of gold particles
with a primary diameter of 34 nm in comparison to that of 4 nm particles (~55 nm and ~18 nm
aerodynamic diameter of agglomerates, respectively). Our studies in mice exploited the availability
8
of gold nanoparticles with narrow size distributions to define more tightly whether a size threshold
is present for extra-pulmonary translocation. Significant translocation into the circulation occurred
only at primary particle sizes below 30 nm, with an inverse association between size and blood
levels below this. Others have shown a size-dependent association for translocation of
nanoparticles into the blood of healthy rats,40 that was balanced with clearance by the kidneys.41
The current studies suggest that clearance from the circulation via the kidney may have a smaller
size threshold (<6 nm),42,43 supporting the data of Balasubramanian et al..39 Thus reliance on
estimates of particle concentrations in the blood alone represents only a ‘snap-shot’ of several
processes (alveolar deposition, epithelial and endothelial cell penetration, particle translocation,
clearance via the kidneys, uptake into other tissues such as the liver, etc) each with their own
conditions for particle passage or retention. Particle surface charge41,35 and protein interactions44,45
will also be important to translocation and clearance processes. Similarly, the ability of the inhaled
particulate to induce pulmonary inflammation could alter translocation rates via increases in
alveolar permeability,46 or raised numbers of alveolar leucocytes that theoretically represent non-
passive carriers to assist translocation. These pathways are the subject of ongoing investigations.
The fate of inhaled nanoparticles that translocate into the circulation remains to be fully
established. Recently, uptake of 7 or 20 nm nanoparticles in the aorta following prolonged
inhalation (15 days) was reported in healthy rats.39 The potential for engineered nanoparticles
(ultra-small superparamagnetic particles of iron oxide, ~30 nm particles) to accumulate at sites of
inflammation, including atherosclerotic plaques, has been considered, but only following
intravenous injection.47 In the present study, we administered nanoparticles via the lung of a well-
validated mouse model of atherosclerosis. Gold was detected in high concentrations in the liver,
confirming the liver as a primary route of clearance following translocation. Importantly,
nanoparticles also accumulated at sites of vascular inflammation, but not in non-atherosclerotic
blood vessels. The translocation and accumulation of gold nanoparticles in atherosclerotic plaque
was confirmed in man. Whilst the small sample size did not permit comparison of the extent of
translocation between patients and healthy controls, Raman microscopy was sufficiently sensitive
9
to detect particulate gold in carotid plaque. The cellular and kinetic mechanisms, as well as the
particle characteristics, that determine the way circulating nanoparticles accumulate with
atherosclerotic plaques require further investigation, although rheological conditions and
inflammatory cells are likely to play a significant role in particle accumulation at sites of disease.
If inhaled nanoparticles are to alter the pathogenesis of atherosclerosis directly then
particles need to access susceptible sites and have the necessary physiochemical properties to
induce toxicity. Our current studies required the use of an inert and identifiable nanoparticle, thus
we could not address the pathobiological consequences of translocated nanoparticles taken up
into atherosclerotic plaques. Indeed, repeated instillation of gold nanoparticles did not increase the
size of atherosclerotic plaques in the carotid of ApoE-/- mice. However, combustion-derived
nanoparticles are known to induce oxidative stress and promote inflammation, and as such, are
likely to directly promote atherosclerosis should they translocate and deposit in the vessel wall.17
We cannot address whether the pro-atherosclerotic effects of exposure to air pollution are due to
accumulation of combustion-derived nanoparticles at sites of vascular inflammation in the present
study, but our observations suggest this is both plausible and likely if nanoparticles were to
accumulate in sufficient numbers. Furthermore, if nanoparticles accumulate in areas of
inflammation elsewhere in the body, this could have important implications for other diseases,48 the
potential for particles to cross the placental barrier during pregnancy,49,50 as well as the progression
of, and theranostics for, cancer.51
These findings have immediate relevance for the nanotechnology industry where a diverse
range of engineered nanomaterials is being developed for an ever-increasing number of
applications. The fate of engineered nanoparticles and effect on health following exposure are
largely unknown,1,2 especially in relation to the cardiovascular system.4 These studies use gold
nanoparticles; a commonly used nanoparticle and one that is being developed for clinical
therapeutics. However, the biokinetics we observe here for gold, may also extend to other
nanomaterials including those with greater surface reactivity. Different classes of nanomaterials
vary greatly in their ability to cause inflammation and cytotoxicity, thus it follows that there will be
marked differences in their impact on health in both occupational settings and in the wider
10
community exposed to nanomaterials. While data is still relatively sparse, a number of studies
suggest that pulmonary exposure to a range of different inhaled nanoparticles may promote
cardiovascular disease.4,52,53 A better understanding of how nanomaterials cross physiological
barriers, and their fate thereafter, will be vital to allow for a safe-by-design approach for new
nanomaterials.
CONCLUSIONS
We demonstrate that nanoparticles translocate from the lung to the circulation and selectively
accumulate at sites of vascular inflammation in both animal models of disease and in man. These
observations suggest that the fate of inhaled nanoparticles, as well as their size, is crucial for their
potential to affect cardiovascular disease adversely. A greater understanding of how nanoparticles
translocate and accumulate at sites of disease is of paramount importance for the risk assessment
of both environmental and engineered nanoparticles.
11
METHODS
Full methods and any associated references are available in the online Supporting Information
supplement.
Clinical Studies
All subjects gave their written informed consent, and the studies were reviewed and
approved by the local research ethics committee according with the Declaration of Helsinki.
Healthy male non-smoking volunteers aged 18-35 (Supplementary Tables S1 & S2) were
exposed to gold nanoparticles for 2 hours via inhalation, during intermittent exercise, in a specially
built human controlled exposure chamber (Supplementary Figure S1A & S1B). Gold
nanoparticles (~5 nm primary diameter) were freshly generated from gold electrodes using a
commercial spark generator (Palas CFG1000, Palas GmbH, Karlsruhe, Germany). In subsequent
experiments larger single spherical particles were generated by extending the residence time in
argon via a growth loop to agglomerate particles, followed by thermal fusing of particles. Following
exposure, volunteers were taken to the adjacent clinical research facility where blood samples
were obtained before and after gold exposure and at predefined intervals. A 24-h urine sample was
also taken, beginning immediately prior to exposure, from an empty bladder.
Patients presenting to the Lothian Stroke services with a recent acute cerebrovascular
syndrome (transient ischaemic attack, amaurosis fugax or stroke) and scheduled for carotid
endarterectomy were also recruited (Supplementary Table S3). Patients were exposed to gold
nanoparticles for two periods of 2-h the day prior to their surgery, but were not requested to
exercise. At surgery, the plaque was excised intact as part of a standard carotid endarterectomy
and patch angioplasty. The specimen was then snap frozen in liquid nitrogen and transferred to a
freezer for subsequent batch analysis.
Animal Studies
All experiments were performed according to the Animals (Scientific Procedures) Act 1986
(UK Home Office).
12
Male apolipoprotein E knockout (ApoE-/-) mice were fed a high-fat (21% fat) “Western diet”
for 8 weeks.15 Gold nanoparticles in suspension (citrate-coated, primary particle size 5nm;
BBInternational, UK; 50 µL of 2.3 mg/mL), or vehicle, were administered to the lungs of mice by
instillation twice weekly for the last 5 weeks of feeding (Supplementary Figure S1C). In
experiments exploring particle size, 2, 5, 10, 30 or 200 nm particles (BBInternational) were used as
above, with the exception that a stock solutions were diluted in order that all sizes were of an
equivalent mass concentration (0.8 mg/mL).
Animals were euthanized ~18 hours after the last instillation. Blood samples from the vena
cava and liver biopsies were taken, and stored in soda lime digestion tubes at -80oC. The lungs
were cannulated via a small incision in the trachea and lavaged 3 times with 0.8 mL sterile saline
to collect bronchoalveolar lavage fluid for measurement of lung inflammation. The aortic arch area
and descending thoracic aorta were cleaned of perivascular fat, rinsed in saline, blotted dry and
frozen at -80oC in digestion tubes. The aortic arch region is used as an area of vasculature with
extensive atherosclerotic lesions of a mixed ‘complex’ plaque phenotype, whereas the descending
aorta is largely free of atherosclerotic plaques in mice at this end under this feeding regime 15.
Quantification of Gold in Biological Samples using HR-ICPMS
Biological samples were completely dissolved in aqua regia (HNO3 and HCl; 120oC) and
diluted with ultrapure water to a final volume of 10 mL before analysis for gold content using high
resolution inductively-coupled plasma mass spectroscopy (HR-ICPMS; Element XR, Thermo
Fisher, Bremen, Germany).54,55 For quality control aspects on the relevant ultra-trace level, two
reference samples “SEROnorm Trace levels in blood” (supplied by C.N. Schmidt, Amsterdam, The
Netherlands; Supplementary Table S4) were analyzed, using two different batch numbers. The
methodological limit of quantification was 0.03 ng/gram of blood, and the coefficient of variation
was 5.5% for blood samples run in triplicate.
Detection and Visualization of Gold Nanoparticles in Biological Tissues
13
Samples of the aortic arch for transmission electron microscopy were fixed for 2-h in 3%
glutaraldehyde and post-fixed for 45 min in 1% osmium tetroxide. Ultrathin sections (60 nm thick)
were cut from selected areas, stained in uranyl acetate and lead citrate, then viewed in a Phillips
CM120 Transmission electron microscope.
Biological samples were fixed in 10% neutral-buffered formalin before histological
processing, and stained with hematoxylin and eosin (lung) or United States Trichrome (arteries).
Atherosclerotic burden was comprehensively assessed, as previously described15. The carotid
artery bifurcation was used for quantify atherosclerotic burden in gold-treated animals as
brachiocephalic arteries were used for detection of gold by HR-ICMPS.
Raman spectroscopy was performed using unstained histological sections that had been de-
paraffinized in graded alcohol. Detection of gold particulate was assisted by conjugation to silver
using a LI-silver enhancement kit (Molecular Probes, Invitrogen, Paisley, UK). Tissue sections
were incubated in 50 nM glycine in phosphate-buffered saline (pH 7.4; 5 min) to block aldehyde
groups in the fixation reagent, then thoroughly rinsed with dH2O to remove all traces of phosphate
ions from PBS. Silver staining reagents were added for 5 min before washing with water and
leaving to air dry.
Detection of gold within tissue samples was performed by Raman spectroscopy (Renishaw
inVia, Renishaw UK). Raman spectra from the lung tissue sections were collected with exposure
time of 60 seconds and 10% laser power, at 514 nm. Spectra were quantified by transforming data
to area under the curve (spectra) between a Raman shift of 950 – 1750 cm-1.
Data Analysis
For parametric data (Gaussian distribution) values reported are mean±SEM with differences
between groups analyzed by analysis of variance (ANOVA) with Bonferroni post-hoc tests, and
Student’s t-tests, where appropriate (GraphPad Prism v6.0a). For non-parametric (non-normally
distributed) data values are reported as median ± interquartile range, with differences between
groups analyzed by Kruskal-Wallis followed by Dunn’s post-hoc tests, and Mann-Whitney tests.
ROUT’s outlier56 (Q=1%, GraphPad Prism v6.0a) was used to detect the presence of outliers in
14
conditions where normalization to very small tissue weights (aortic arch and descending aorta)
skewed data sets. P<0.05 was taken as statistically significant.
15
ASSOCIATED CONTENT
A single file named “MILLER_Supplement” has been submitted containing the following
supporting information:
Supplementary Tables S1-S4
Supplementary Figures S1-S4
Methods in Full
CORRESPONDING AUTHOR
Dr Mark Miller
University/BHF Centre for Cardiovascular Science
University of Edinburgh
Queens Medical Research Institute
47 Little France Crescent
Edinburgh, EH16 4TJ
United Kingdom
Tel: +44 131 242 9334
Fax: +44 131 242 9215
Email: [email protected]
16
AUTHOR CONTRIBUTIONS
The manuscript was written through contributions of all authors. All authors have given
approval to the final version of the manuscript.
* Joint and equal contribution to first authorship
# Joint and equal contribution to senior authorship
MRM, JR, JPL, PWFH, AV, KD, DEN, RD and NLM conceived and designed the studies. MRM,
JR, SGM, and RD performed the animal studies and collected data. JPL and SW performed the
clinical studies. ATV performed the studies in carotid endarterectomy patients. PK performed the
HR-ICPMS analysis. PS, JR, SC and CJC performed the Raman Spectroscopy. FRC, PHBF and
AJFB generated the gold exposures for the clinical studies. JPL, MRM, JBR and NLM performed
data analysis. MRM, JPL and NLM drafted the manuscript and all authors reviewed and approved
the final manuscript.
FUNDING SOURCES
British Heart Foundation Programme Grant (RG/10/9/28286)
British Heart Foundation Project Grants (PG/10/042/28388, PG/12/8/29371)
British Heart Foundation Senior Research Fellowship (FS/16/14/32023)
British Heart Foundation Special Project Grant (SP/15/8/31575)
British Heart Foundation Chair Award (CH/09/002)
Colt Foundation Project Grant (CF/07/11)
Dutch Ministry of Infrastructures and Environment (M/630196)
Department of Health (UK) Project Grant (PR-NT-0208-10025).
17
ACKNOWLEDGMENTS
We would like to thank S. McSheaffrey and J. Sethunarayanan for their assistance with the
clinical studies, as well as the nursing staff at the Clinical Research Facility, Royal Infirmary of
Edinburgh which is supported by NHS Research Scotland (NRS) through NHS Lothian. We thank
S. Mitchell for his technical assistance with the electron microscopy and E. Miller for her assistance
with the histological analysis. Thanks also to B. Stolpe at University of Birmingham Facility for
Environmental Nanoscience Analysis and Characterisation (FENAC) for his help with the
characterization of the gold nanoparticles. This research was supported by Programme and Project
Grants from the British Heart Foundation (RG/10/9/28286; PG/10/042/28388; PG/12/8/29371) and
a Project Grant (202111) from the COLT Foundation, with additional support from the Dutch
Ministry of Infrastructures and Environment (M/630196) and the UK Department of Health (PR-NT-
0208-10025). MRM, NLM and DEN are supported by a Special Project Grant (SP/15/8/31575), the
Butler Senior Clinical Research Fellowship (FS/16/14/32023) and a Chair Award (CH/09/002),
respectively, from the British Heart Foundation. DEN is the recipient of a Wellcome Trust Senior
Investigator Award (WT103782AIA).
ABBREVIATIONS
99mTc = 99m technetium; ANOVA = analysis of variance; ApoE-/- = Apolipoprotein-E knockout
mouse; HR-ICPMS = high resolution inductively-coupled plasma mass spectrometry;
18
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22
FIGURES
Graphical Abstract, version 1
Graphical Abstract, Version 2
23
Figure 1. Gold nanoparticle translocation in man. (A) Representative transmission electronmicrograph showing gold particles generated by the spark generator with a primary particle size of3.8 nm in loose agglomerates (B) with an aerodynamic count median diameter of 18.7 nm(geometric standard deviation = 1.5). Abbreviations: AFM = atomic force microscopy; DLS =dynamic light scattering; SMPS = scanning mobility particle sizer; TEM = transmission electronmicroscopy. (C) In man, gold was detectable in the bloodstream in some subjects within 15 mins ofthe 2-hour exposure, and was detectable in the majority of subjects at 24 hours (12/14 subjects,86%). (D) Quantification of gold concentrations in blood and urine. Blood concentrations wereclose to the limit of quantification (0.03 ng/gram of blood; dotted line) at early time points. Greysymbols represent values below the limit of quantification, but above the limit of detection (0.01ng/gram of blood). Data expressed as median ± interquartile range.
24
Figure 2. Effect of particle size on translocation. (A) Representative transmission electronmicrograph showing gold particles (4 nm primary diameter) generated by the spark generator (i)with and (ii) without particle fusing to obtain large (~34 nm primary diameter) particulates from thesecond clinical exposure study. (B) Characteristics for aerosolized gold particle exposures inclinical studies (C) Levels of gold detectable in the bloodstream in subjects exposed to differentsizes of gold particles. (D) Levels of gold detectable in the urine of subjects exposed to differentsizes of gold particles. (E) Levels of gold in the blood of mice exposed to 5 weeks repeatedpulmonary instillation of different sizes of gold nanoparticles (F) murine-liver. (G) murine urine.Data expressed as mean±SEM, unless otherwise stated.
25
Figure 3. Accumulation of gold nanoparticles in tissues of ApoE-/- mice following pulmonaryinstillation. (A) Transmission electron micrograph of gold particles used for instillations in mice,with a primary particle size of 5 nm and a median agglomerate size of 7.8 nm. (B) Instillation ofgold nanoparticles caused mild lung inflammation, measured as total cell count in BALF.Mean±SEM., ***P<0.001, unpaired t-test, n=6. (C) Gold was detectable in the blood and liver ofgold-treated apolipoprotein-E knockout (ApoE-/-) mice. (D) Gold particles accumulated in areas ofthe vasculature rich in atheroma (aortic arch) compared to those free of plaque (descending aorta).Mean±SEM, n=7-8 (preclinical study). *P<0.05, ***P<0.001, Mann Whitney test compared tovehicle-treated animals, †P<0.05, Mann Whitney test compared to aortic arch of gold-treated mice.
26
Figure 4. Visualization of gold nanoparticles in the lung and atherosclerotic plaques ofApoE-/- mice. (A) Gold particles were clearly visible within lungs of gold-treated animals, largelywithin alveolar macrophages. (B) Computational coloring to illustrate capillaries (red), alveolarspaces (blue) and gold particles (yellow). (C) Section of atherosclerotic plaque from gold-treatedmouse showing a distinctive cluster of red-purple granules within a macrophage-derived foam cell.(D) Transmission electron micrograph of atherosclerotic plaque within the aortic arch. Insets;examples of particle clusters within the plaques of gold-treated mice. med=vascular media,pla=atherosclerotic plaque, lumen=blood vessel lumen, fc=foam cell, lc=lipid core.
27
Figure 5. Raman spectroscopy to confirm the presence of particulate gold in lung andatherosclerotic plaques of ApoE-/- mice. (A) Suspensions of gold nanoparticles followingconjugation with silver produced a unique Raman spectra (red), that was not seen with either gold(blue) or silver alone (green). (B) Representative Raman spectra from silver-augmented gold insections of lung (red) and atherosclerotic plaques (orange) from a gold-treated mouse, not seen inlung (blue) or plaque (green) sections from a vehicle-treated mouse. Raman spectra from (C) lungand (D) atherosclerotic plaques; Data expressed as median ± interquartile range. ***P<0.001,Mann Whitney test comparing gold-treated (n=64 grids from 3 mice) to vehicle-treated (n=39 gridsfrom 2 mice) mice.
28
Figure 6. Gold nanoparticle exposures in patients undergoing carotid endarterectomy. (A)Isolation of atherosclerotic plaque from the carotid artery. (B) Overlaid representative Ramanspectroscopy spectra: black = silver stained spark-generated gold nanoparticles on glass slide (notissue), blue = silver-stained endarterectomy sample from non-exposed patient, red =silver-stainedendarterectomy sample from gold-exposed patient. (C) Visualisation of gold in endarterectomysamples using heat-map of Raman intensity. Highlighted box within tissue denotes scanned area,with blue-green color representing baseline intensity (no gold) and red color showing high Ramanintensity (gold particulate).
29
SUPPLEMENTARY MATERIAL
Supplementary Table S1. Baseline characteristics of healthy volunteers
participating in single size (5 nm) gold exposure study
Parameter All subjects (n=14)
Age, years 22 (19 – 34)
Height, cm 180 ± 4
Weight, kg 79 ± 11
Body Mass Index, kg/m2 24.3 ± 2.7
Body Surface Area, m2 2.76 ± 1.28
FEV1, L 4.7 ± 0.4
Vital capacity, L 5.6 ± 0.5
Systolic blood pressure, mmHg 130 ± 12
Diastolic blood pressure, mmHg 74 ± 10
Heart rate, bpm 74 ± 15
Data expressed as mean ± standard deviation or median (range) as appropriate. FEV 1 = forced expiratoryvolume in 1 second. Body surface area calculated using the Mosteller formula1.
30
Supplementary Table S2. Baseline characteristics of healthy volunteers
participating in size comparison (5 vs 30 nm) gold nanoparticle exposure study
ParameterSmall particle
exposures (n=10)Large particle
exposures (n=9)
Age, years 28 ± 13 22 ± 4
Height, cm 177 ± 5 178 ± 9
Weight, kg 74 ± 11 79 ± 13
Body Mass Index, kg/m2 24 ± 3 25 ± 3
Heart rate, bpm 70 ± 9 75 ± 15
Systolic blood pressure, mmHg 131 ± 7 145 ± 15
Diastolic blood pressure, mmHg 75 ± 9 79 ± 9
Data expressed as mean ± standard deviation
31
Supplementary Table S3. Baseline characteristics of carotid endarterectomy
patients
Parameter Controls (n = 10) Gold exposed (n=3)
Age, years 72 (62 – 76) 69 (56 – 79)
Sex, men (%) 7 (70) 2 (67)
Height, cm 165 ± 10 169 ± 1
Weight, kg 73 ±12 93 [92 – 112.6]
Body Mass Index, kg/m2 27.0 ± 4.4 32 [32 – 40]
Systolic Blood Pressure,mmHg
151 ± 22 147 ± 18
Diastolic Blood Pressure,mmHg
81 ± 13 78 ± 7
Creatinine, µmol/L 82.7 ± 28.7 75.7 ± 11.1
Data expressed as mean ± standard deviation, mean (range) or median [interquartile range] as appropriate.
32
Supplementary Table S4. Levels of gold in reference samples for HR-ICPMS
Reference sample (Lot number)
SEROnormconcentrations
Laboratoryconcentrations
Sample 1 (201605; 503109) 0.022±0.003 ng/mL 0.017±0.005 ng/mL
Sample 2 (201205; 1003192) 0.007±0.002 ng/mL 0.009±0.006 ng/mL
Data expressed as mean ± standard deviation
33
Supplementary Figure S1. Experimental design. (A) Protocol and sampling procedure for
the gold nanoparticle inhalation studies in man. (B) Exposure monitoring equipment and
volunteer wearing inhalation apparatus. Image has been modified to protect the identity of
the volunteer. (C) Feeding and instillation protocol for experiments with gold nanoparticle-
treated apolipoprotein E knockout mice. (D) Regions of the vasculature selected to
investigate the vascular uptake of translocated nanoparticles in atherosclerotic (aortic arch
region) and non-atherosclerotic (descending aorta) arteries from mice using the 8-week
feeding regime.
34
Supplementary Figure S2. Use of transmission electron microscopy to visual
translocated gold nanoparticle within atherosclerotic plaques. (A) Granular electron-dense
particle clusters were evident in some section of tissue from the lipid-rich areas of plaques.
(B) Other electron dense structures were also seen in the plaques of both saline- and
gold-treated animals. However, these regions had a layered structure likely representing
calcium bound to lipid droplets or membranous segments from necrotic cell nuclei.
35
Supplementary Figure S3. Raman spectroscopy consistently confirms the presence of
particulate gold in tissue sections of gold-treated animals. Characteristic spectra of gold-
silver conjugates (see main manuscript) were observed in the (A) lung and (B)
atherosclerotic plaques in different gold-instilled animals, but not in saline-instilled animals,
using a subset of tissues from 5 different animals.
36
Supplementary Figure S4. HR-ICPMS measurement of gold in the urine and blood of
endarterectomy patients. Data expressed as mean ± S.E.M. (n=10 non-exposed, n=3
gold-exposed).
37
Methods in Full
Gold nanoparticles
In clinical studies, gold nanoparticles (~5nm primary diameter) were freshly generated from gold
electrodes (3-4 mm thick, 6mm diameter) using a commercial spark generator operated at a fixed
spark frequency (Palas CFG1000, Palas GmbH, Karlsruhe, Germany) in an atmosphere of pure
argon, prior to dilution in filtered and conditioned air for exposures. Larger gold nanoparticles for
clinical inhalation studies were generated by extending the residence time in argon via a through a
growth loop, consisting of coiled tubing and buffers, allowing particles to agglomerate by
coagulation. The agglomerates were subsequently fused into spherical gold particles of about 30
nm primary diameter by passing the gold aerosol through an oven heated to 600oC, before being
diluted in filtered air, which resulted in solid spherical single particles. Exposure was monitored in
real time for particle number concentration (CPC 3022, TSI Inc., St Paul, USA), surface area
(NSAM 3550, TSI Inc., St Paul, USA) and size distribution (SMPS 3080, TSI Inc., St Paul, USA).
Samples were collected onto two parallel Teflon filters (R2PJ047, Pall Corp., Ann Arbor, USA) and
gravimetrically analyzed for particle mass concentration. Primary particle size in the resulting
suspension was determined by transmission electron microscopy (TEM; Phillips CM120
Transmission electron microscope; FEI UK Ltd, Cambridge, UK) and atomic force microscopy
(AFM; XE-100 AFM, Park Systems Corp., Korea)2.
Gold nanoparticle suspensions used for the murine study were purchased from
BBInternational (Cardiff, UK). TEM (Phillips CM120; FEI UK Ltd) was used to confirm the primary
particle size. Mean particle size in suspension was determined by dynamic light scattering to
ensure there was minimal agglomeration of gold particles prior to instillation. Suspensions were
diluted 100-fold in PBS (BBInternational) and particle size measured using a PS90 Particle Size
Analyzer (Brookehaven Instrument Corporation, New York, USA) set to the following parameters:
37oC, angle 90o; run duration 1 min; refractive index of particles 0.2 ‘real’ (0 ‘imaginary’); dust cut-
off 30.
38
Clinical Studies
All subjects gave their written informed consent, and the studies were reviewed and approved by
the local research ethics committee according with the Declaration of Helsinki.
Healthy volunteer studies
Healthy male non-smoking volunteers aged 18-35 (Supplementary Tables S1 & S2) were
recruited from the University of Edinburgh. Subjects taking regular medication and those with
clinical evidence of atherosclerotic vascular disease, arrhythmias, diabetes mellitus, hypertension,
renal or hepatic failure, asthma, significant occupational exposure to air pollution, or an intercurrent
illness likely to be associated with inflammation were excluded from the study. Subjects had
normal lung function and reported no symptoms of respiratory tract infection for at least 6 weeks
before or during the study. All subjects abstained from alcohol for 24 hours and from food, tobacco,
and caffeine-containing drinks.
Healthy subjects attended for a screening visit where lung function was performed
(Vitalograph, Buckingham, UK) and a cardiopulmonary exercise carried out to confirm the workload
needed to generate an average minute ventilation of 25 L/min/m2 body surface area. Subjects were
exposed to the gold aerosol for 2 hours during intermittent exercise on a recumbent exercise
bicycle (Heinz-Kettler GmbH, Ense-Parsit, Germany) at the predefined workload, in a specially built
human controlled exposure chamber. The output of the nanoparticle generator was mixed with
HEPA filtered air at 80 L/min, stabilized in a 200 L mixing chamber (residence time ~2.5 min) and
delivered to the subject through a facemask (ComfortFull 2, Philips Healthcare, Best, Netherlands)
(Supplementary Figure S1A & S1B). Following exposure, volunteers were taken to the adjacent
clinical research facility, for sample collection were carried out in a quiet, temperature-controlled
39
room maintained at 22°C to 24°C with subjects lying supine. Subjects remained indoors during this
time to minimize additional exposure to particulate air pollution.
Carotid endarterectomy studies
Patients presenting to the Lothian Stroke services with a recent acute cerebrovascular syndrome
(transient ischaemic attack, amaurosis fugax or stroke) and scheduled for carotid endarterectomy
were recruited (Supplementary Table S3). Exclusion criteria were: recent body-piercing or history
of gold tooth filling, psychiatric illness/social situations that would limit compliance with study
requirements, patients with new stroke and a modified Rankin score >3, pregnant women,
participation in the study would result in delay to carotid surgery, history of allergic reaction
attributed to elemental gold or molecules including gold. The study followed an accepter/rejecter
model (i.e. patients who didn’t consent to gold inhalation, could be included as controls – a
randomized model was not felt to be feasible given the significant scheduling challenges in these
acutely unwell patients who require surgery as soon as possible).
Gold exposed patients inhaled gold nanoparticles (~5 nm diameter) on the day prior to
surgery, as described above. Patients were not exercised during exposure (to minimize the risk of
mechanical stress on a fragile and acute plaque), thus, the duration of exposure was increased to
two periods of 2-h (first in morning, the second in the afternoon) to maximize particle exposure.
Blood and urine samples were taken prior to gold exposure and at the time of surgery. For control
patients blood and urine samples were only taken at the time of surgery. At surgery, the plaque
was excised intact as part of a standard carotid endarterectomy and patch angioplasty. The plaque
was immediately handed off table, oriented and photographed. The specimen was then snap
frozen in liquid nitrogen and transferred to a freezer for subsequent batch analysis.
Blood and urine sampling
40
Blood samples were obtained through an in-dwelling intravenous cannula before and after gold
exposure and at predefined intervals (Supplementary Figure S1A). Samples were collected into
Lithium Heparin (S-Monovette® for trace metal analysis, Sarstedt AG, Nümbrecht, Germany)
before being transferred to disposable soda lime digestion tubes (Duran Group, Mainz, Germany)
and stored at -20oC before further analysis. Urine was collected over 24 hours following exposure.
Total urine was thoroughly mixed and subsamples were separated into aliquots and frozen at
-40oC until further analysis. Blood samples collected before and 24 hours after exposure to gold
nanoparticles, as well as later time points in some studies, and were analysed in the regional
hematology and clinical chemistry reference laboratory.
Animal Studies
All experiments were performed according to the Animals (Scientific Procedures) Act 1986 (UK
Home Office).
Animals and exposures
Male apolipoprotein E knockout (ApoE-/-) mice (Apoetm1Unc/J; Charles River UK; n=8/group) aged
12-14 weeks at the start of the protocol were fed a high-fat “Western diet” (Research Diets, New
Brunswick, USA) for 8 weeks3. Gold nanoparticles (citrate-coated, primary particle size 5nm;
BBInternational, UK) in suspension (50 µL of 2.3 mg/mL), or vehicle, were sonicated for 20 min
then administered to the lungs of mice by instillation twice weekly for the last 5 weeks of feeding
(Supplementary Figure S1C). In experiments exploring particle size, 2, 5, 10, 30 or 200 nm
particles (BBInternational) were used as above, with the exception that a stock solutions were
diluted in order that all sizes were of an equivalent mass concentration (0.8 mg/mL).
41
Animals were euthanized ~18 hours after the last instillation. Blood samples from the vena
cava and liver biopsies were taken, and stored in soda lime digestion tubes at -80oC. The lungs
were cannulated via a small incision in the trachea and lavaged 3 times with 0.8 mL sterile saline
to collect bronchoalveolar lavage fluid (BALF). BALF was centrifuged at 180g for 5 min at 4°C, the
supernatant was removed, and the cell pellets combined. Cell pellets were resuspended in 1 mL of
phosphate-buffered saline (PBS) and total cell numbers were counted using an automatic cell
counter (Satorius Steadman, Chemometec, Nucleocounter®, Gydevang, Denmark). The aortic arch
(and immediate branches: ~8 mm of brachiocephalic, left carotid and left subclavian arteries) and
descending thoracic aorta (from end of curvature of arch to intersection with diaphragm) were
cleaned of perivascular fat, rinsed in saline, blotted dry and frozen at -80oC in digestion tubes. The
aortic arch region is used as an area of vasculature with extensive atherosclerotic lesions of a
mixed ‘complex’ plaque phenotype4, whereas the descending aorta is largely free of atherosclerotic
plaques in mice at this end under this feeding regime (Supplementary Figure S1D)3. Sample
weight was determined by comparison of tubes weights before and after collection of the sample.
Quantification of Gold in Biological Samples using HR-ICPMS
Biological samples were completely dissolved in aqua regia (0.5 mL 60% HNO3 and 1.5 mL 30%
HCl; 120oC) and diluted with ultrapure water to a final volume of 10 mL before analysis for gold
content using high resolution inductively coupled plasma mass spectroscopy (HR-ICPMS; Element
XR, Thermo Fisher, Bremen, Germany)5, 6. Urine samples were directly diluted with aqua regia by a
factor of 10 and analysed afterwards by HR-ICPMS. An external calibration curve of the
interference-free isotope 197Au was performed in the low-resolution mode between 0 and 40 ng/L.
On-line addition and correction with an internal standard (1 µg/L rhodium with the measured
isotope 103Rh in the low-resolution mode) and the correction for the chemical blank were applied
too. All chemicals used were of analytical grade or of high purity. Nitric acid (HNO3) and
hydrochloric acid (HCl) were purchased from Merck, Darmstadt, Germany. The calibration
standard solution of gold (Au), as well as a solution of the element rhodium (Rh) used as internal
42
standard, were prepared using single element stock solutions with a concentration of 1000 mg/mL
(both Inorganic Ventures; Instrument Solutions, Nieuwegein, The Netherlands). For quality control
aspects on the relevant ultra-trace level, two reference samples “SEROnorm Trace levels in blood”
(supplied by C.N. Schmidt, Amsterdam, The Netherlands; Supplementary Table S4) were
analyzed, using two different batch numbers. The methodological limit of quantification was 0.03
ng/gram of blood, and the coefficient of variation was 5.5% for blood samples run in triplicate.
Histology and electron microscopy
Additional animals (3 gold-instilled, 2 vehicle-instilled) were used to provide arterial
(brachiocephalic) and pulmonary tissue for histology and electron microscopy. The lungs of these
animals were not lavaged and were instead perfusion fixed with ~2 mL of methacarn fixative (60%
methanol, 30% chloroform and 10% glacial acetic acid) and the lungs allowed to slowly inflate
under gravity. Arterial and liver samples were fixed overnight in 10% neutral-buffered formalin
before being transferred to 50% ethanol until time of analysis. Samples were embedded in paraffin,
5 μm thick sections prepared, and then stained with hematoxylin and eosin (lung) or United States
Trichrome (arteries).
Samples of the aortic arch for transmission electron microscopy (TEM) were fixed for 2-h in
3% glutaraldehyde (in 0.1 M sodium cacodylate buffer, pH 7.3) and post-fixed for 45 min in 1%
osmium tetroxide (in 0.1 M sodium cacodylate). The samples were then dehydrated in increasing
concentrations of acetone before being embedded in Araldite® resin. Sections (1 μm thick) were
cut on a Reichert OMU4 ultramicrotome (Leica Microsystems Ltd, Milton Keynes, UK), stained with
toluidine blue and viewed in a light microscope to select suitable areas for investigation. Ultrathin
sections (60 nm thick) were cut from selected areas, stained in uranyl acetate and lead citrate,
then viewed in a Phillips CM120 Transmission electron microscope (FEI UK Ltd, Cambridge, UK).
Images were taken on a Gatan Orius CCD camera (Gatan Oxon, UK).
43
Silver Staining and Raman Spectroscopy
Raman spectroscopy was performed using unstained histological sections that had been de-
paraffinized in graded alcohol. Detection of gold particulate was assisted by conjugation to silver
using a LI-silver enhancement kit (Molecular Probes, Invitrogen, Paisley, UK). Tissue sections
were incubated in 50 nM glycine in phosphate-buffered saline (PBS pH 7.4; 5 min) to block
aldehyde groups in the fixation reagent, then thoroughly rinsed with dH2O to remove all traces of
phosphate ions from PBS. Silver staining reagents were added for 5 min before washing with water
and leaving to air dry.
Detection of gold within tissue samples was performed by Raman spectroscopy (Renishaw
inVia, Renishaw UK). The laser was set at 514 nm (based on absorbance spectra from preliminary
experiments) with pinhole at 15 mW. Incident light was focused to the samples by 50× Olympus
long working distance objective (NA = 0.45) with 2.1 µm focal diameter. Exposure time and laser
power used in the determination were varied depending on tissue sections. Raman spectra from
the lung tissue sections were collected with exposure time of 60 seconds and 10% laser power. In
arterial sections the exposure time of 100 seconds and 50% laser power were used. The charge
coupled device (CCD) detector was used with Peltier cooling to -70˚C, using a CCD area of 9,000-
11,000 counts. In carotid samples, a rectangular selection of 5,000-6,000 scans was performed to
generate a ‘heat map’ for the localization of spectra positive for gold.
Raman spectra were processed using Wire2.0 software and analyzed using Origin8.0
software. Baselines of all Raman spectra were corrected to a horizontal slope in order to calculate
noise intensities. Range of frequency with no Raman signal was used to determine the intensities
of noise by taking the average signal + (3x standard deviation). Intensities of signal were
calculated by dividing the intensity of signal by the intensity of noise. Spectra were quantified by
transforming data to area under the curve (spectra) between a Raman shift of 950 – 1750 cm-1.
44
Quantification of atherosclerosis
Atherosclerotic burden was comprehensively assessed, as previously described3. The aortic arch
and descending thoracic aorta was cut longitudinally and lipid-rich atherosclerotic plaques were
stained en-face using Sudan IV. The coverage of the aortic surface with red-stained plaques was
quantified using Image-J software.
The brachiocephalic arteries was selected for histological analysis of plaque burden as this
region of artery develops ‘complex’ fibroelastic plaques that are representative of human plaque
structure7 and undergo extensive expansion and remodeling during the instillation period of the
protocol8, 9. Arteries were fixed in formalin and histological sections were taken in triplicate at 100
m intervals, beginning at the first section of artery with a fully intact media. Sections were stained
with United States Trichrome (UST). The cross-sectional area of the plaque was measured and
standardised to the area of the vascular media. A single mean value of atherosclerotic burden for
each animal was calculated from the plaque size from each complete serial section throughout the
brachiocephalic artery. The carotid artery bifurcation was used for quantify atherosclerotic burden
in gold-treated animals as brachiocephalic arteries were used for detection of gold by ICMPS.
Plaque structural complexity was assessed by counting distinct adjoining or overlying
plaques within each section10. Quantitative measurement of plaque constituents was performed
using single UST-stained sections from the region of maximal plaque growth. Images of sections
were imported into Adobe Photoshop v11.0, and a colour range was selected from three randomly
chosen positively stained sections, which was then used to identify positively stained plaque
components from all subsequent slides.
Data Analysis
For parametric data (Gaussian distribution) values reported are mean±SEM with
differences between groups analyzed by analysis of variance (ANOVA) with Bonferroni post-hoc
45
tests, and Student’s t-tests, where appropriate (GraphPad Prism v6.0a). For non-parametric (non-
normally distributed) data values are reported as median ± interquartile range, with differences
between groups analyzed by Kruskal-Wallis followed by Dunn’s post-hoc tests, and Mann-Whitney
tests. ROUT’s outlier11 (Q=1%, GraphPad Prism v6.0a) was used to detect the presence of outliers
in conditions where normalization to very small tissue weights (aortic arch and descending aorta)
skewed data sets. P<0.05 was taken as statistically significant.
46
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