Biodistribution of single and aggregated gold nanoparticles exposed to the human lung epithelial tissue barrier at the air-liquid interface
Durantie et al. Particle and Fibre Toxicology
Biodistribution of single and aggregated gold nanoparticles exposed to the human lung epithelial tissue barrier at the air-liquid interface
Estelle Durantie 0 2
Dimitri Vanhecke 0 2
Laura Rodriguez-Lorenzo 0 2
Flavien Delhaes 0 2
Sandor Balog 0 2
Dedy Septiadi 0 2
Joel Bourquin 0 2
Alke Petri-Fink 0 1 2
Barbara Rothen-Rutishauser 0 2
0 BioNanomaterials Group, Adolphe Merkle Institute, Université de Fribourg , Chemin des Verdiers 4, 1700 Fribourg , Switzerland
1 Chemistry Department, University of Fribourg , Chemin du Musée 9, 1700 Fribourg , Switzerland
2 BioNanomaterials Group, Adolphe Merkle Institute, Université de Fribourg , Chemin des Verdiers 4, 1700 Fribourg , Switzerland
Background: The lung represents the primary entry route for airborne particles into the human body. Most studies addressed possible adverse effects using single (nano)particles, but aerosolic nanoparticles (NPs) tend to aggregate and form structures of several hundreds nm in diameter, changing the physico-chemical properties and interaction with cells. Our aim was to investigate how aggregation might affect the biodistribution; cellular uptake and translocation over time of aerosolized NPs at the air-blood barrier interface using a multicellular lung system. Results: Model gold nanoparticles (AuNPs) were engineered and well characterized to compare single NPs with aggregated NPs with hydrodynamic diameter of 32 and 106 nm, respectively. Exposures were performed by aerosolization of the particles onto the air-liquid interface of a three dimensional (3D) lung model. Particle deposition, cellular uptake and translocation kinetics of single and aggregated AuNPs were determined for various concentrations, (30, 60, 150 and 300 ng/cm2) and time points (4, 24 and 48 h) using transmission electron microscopy and inductively coupled plasma optical emission spectroscopy. No apparent harmful effect for single and aggregated AuNPs was observed by lactate dehydrogenase assay, nor pro-inflammation response by tumor necrosis factor α assessment. The cell layer integrity was also not impaired. The bio-distribution revealed that majority of the AuNPs, single or aggregated, were inside the cells, and only a minor fraction, less than 5%, was found on the basolateral side. No significant difference was observed in the translocation rate. However, aggregated AuNPs showed a significantly faster cellular uptake than single AuNPs at the first time point, i.e. 4 h. Conclusions: Our studies revealed that aggregated AuNPs showed significantly faster cellular uptake than single AuNPs at the first time point, i.e. 4 h, but the uptake rate was similar at later time points. In addition, aggregation did not affect translocation rate across the lung barrier model since similar translocation rates were observed for single as well as aggregated AuNPs.
Aggregate; Gold nanoparticle; Air liquid interface cell exposure; Biodistribution; Human epithelial airway model; Translocation; Cellular uptake
Agglomeration and/or aggregation is an ubiquitous
phenomenon observed for nanoparticles (NPs), however,
the interaction of NP agglomerates with cells/tissues
have only rarely being studied, consequently very little is
known on their interaction with biological systems and
subsequent fate [
]. Agglomerates and aggregates are
secondary entities in which single NPs, or primary
particles, are held together. In agglomerates, primary
particles are assembled by weak physical interactions (i.e. van
der Waals forces) and the whole process is reversible,
while aggregates are defined as comprising strongly
bonded primary particles, and the process is irreversible
]. Agglomerates and aggregates will be simplified to the
aggregates term from now on. These assembled NPs
systems display more complex physicochemical properties
than single NPs as their size, morphology, surface area and
effective density will depend additionally on the fractal
dimension and packing factors [
NPs, are major contributors of aggregates in the airborne
ambient air and, have been associated to adverse health
]. During the combustion process, i.e. diesel or
gasoline engines, unburned or partially burned fuel undergo
nucleation process forming single particles with diameter of
about 10–30 nm [
]. These single particles can further
collapse to form aggregates with mean diameter below
100 nm up to several hundreds of nm which results in a
reduced concentration number [
6, 12, 13
Humans are constantly exposed to airborne particles
of different sources in the environment which enter the
human body mainly by inhalation. NPs with a diameter
from 5 to 500 nm can enter and penetrate into the
alveolar region of the lung by diffusion processed [
the deposited NPs have been shown to translocate across
the air-blood barrier reaching the blood or lymphatic
circulation, to be further distributed to secondary organs
]. Evidence suggest that NPs’ translocation in
healthy lungs most likely occurs via transcellular rather
than paracellular pathway [
]. Moreover, findings
support active processes, e.g. endocytotic uptake
mechanism, to be preferentially involved, albeit passive diffusion
is not excluded. It has been shown that NPs’
physicochemical properties, such as size, shape and surface,
influence their uptake into cells and transport across the
lung barrier (translocation) [
]. Some studies in rats
have reported a higher translocation rate of smaller NPs
17, 19, 22, 23
], while in others studies NP surface
charge was found to influence the translocation .
Although aggregation is a common phenomenon, most
of the in vitro and in vivo studies assume NPs remain in
a single state when studying interactions with cellular,
tissue or organ structures. There are only few
investigations explicitly on the interactions of aggregates with
biological systems (for a review see ref. [
]). Among the
very rare in vivo studies, the effect of the aggregate size
or effect of the primary particle size were explored in
rats. It has been shown that smaller aggregates, 20 vs
80 nm, containing same primary single iridium NPs
(2.4 nm) [
] or smaller primary particle size AuNPs, 7
vs 20 nm, forming aggregates with peak diameter of
45 nm [
] exhibited higher translocation and wider
distribution to secondary organs. However, systematic
in-depth studies at the mechanistic level comparing
single particles with aggregates are still missing.
In the present work, the behavior of single and
aggregated AuNPs was compared regarding their
biodistribution across the air-blood tissue barrier by investigating
their cellular uptake and translocation at different time
points (i.e. 4, 24 and 48 h). Well-defined single and
aggregated particles were used as model particles with
hydrodynamic diameters of 32 and 106 nm, respectively,
composed of primary AuNPs of 14.5 nm (core diameter)
stabilized with polymer mixture consisting of polyvinyl
alcohol and polyallyl amine (PVA/PAAm). To simulate a
realistic inhalation the NPs were deposited at the
airliquid interface onto an in vitro 3D human alveolar
epithelial barrier. The 3D human lung model, developed by
Rothen-Rutishauser et al. [
], is composed of human
lung alveolar cells (A549 cell line), primary
humanmonocyte derived macrophages and dendritic cells. The
cells were exposed to single and aggregated AuNPs at
the air-liquid interface at four different concentrations,
i.e. 30, 60, 150 and 300 ng/cm2, and the deposition was
thoroughly characterized by inductively coupled plasma
optical emission spectrometry (ICP-OES) and
transmission electron microscopy (TEM). Cytotoxicity,
proinflammation and cell layer integrity were assessed at 4,
24 and 48 h after exposure. NPs cellular uptake and
translocation were then assessed by measuring the mass
of gold by ICP-OES in the individual compartments (i.e.
apical side, inside the tissue and in the basal medium).
In addition, localization of intracellular NPs was
analyzed by TEM.
Single and aggregated AuNPs were prepared following
the procedure of Hirsch et al. with an adaptation of the
polymer coating [
Synthesis of tiopronin-coated AuNPs: Briefly, to a
solution of tetrachloroauric acid (500 mL, 0.5 mM; Sigma
Aldrich Chemie GmbH, Buchs, Switzerland) in ultrapure
water (MilliQ H2O, Merck Millipore) heated at reflux was
added quickly a warmed solution of sodium citrate (25 mL,
1% w/v) and stirred for 15 min. The reaction mixture was
cooled to room temperature and a solution of tiopronin
(2-mercaptopropionylglycine; Sigma-Aldrich) (15.5 mL,
0.5 mM) was added. The reaction mixture was stirred at
room temperature overnight.
Preparation of polymer mixture PVA/PAAm-(17 kDa)
and PVA/PAAm-(65 kDa): The mixtures of poly(vinyl
alcohol) (PVA) and poly(allylamine) (PAAm),
PVA/PAAm(17 kDa) and PVA/PAAm-(65 kDa) were prepared by
dissolving PVA (11.8% w/v; 5.9 g; 14 kDa, Mowiol 3–85,
Omya AG, Switzerland) and PAAm 17 kDa (0.2% w/v;
490 μL; Fluka solution 20% wt) or PAAm 65 kDa (0.2%
w/v; 980 μL; Fluka solution 10% wt), respectively, in
MilliQ H2O (final volume 50 mL) and stirred overnight.
In the polymer mixtures PVA/PAAm (11.8:0.2, mass
ratio), the PAAm size was changed depending if it was
used to coat single or aggregated AuNPs. The PAAm
65 kDa was chosen to ensure the electrosteric stabilization
of the assembly while PAAm 17 kDa was used to coat
single AuNPs, hence avoiding any aggregation due to
polymer length. However, to make comparative study of
the two systems, it is important to note that amount of
amines remains equal in the two mixtures.
Preparation of single AuNPs: The solution of
tiopronincoated AuNPs (100 mL) was added dropwise to the
aqueous polymer mixture PVA/PAAm-(17 kDa) (9 mL) and
stirred for 4 h at room temperature. After 1 day of storage
at 4 °C, the suspension was centrifuged at 10000 × g for
1 h and the supernatant was collected and centrifuged
again under the same conditions. This process was
repeated one more time.
Preparation of aggregates AuNPs: The solution of
tiopronin-coated AuNPs (10 mL) was treated with HCl
(1 M, 36 μL) so that the mixture reaches a pH of 3. An
aqueous mixture of polymer PVA/PAAm-(65 kDa) was
added to stabilize the agglomerates. After 1 day of
storage at 4 °C, the suspension was centrifuged at 5000 × g
for 1 h. This process was repeated one more time.
UV-Vis spectra of the single and aggregated AuNPs were
recorded in MilliQ H2O using a Jasco V-670
spectrophotometer (Jasco Europe S.R.L., Milano, Italy) with 10 mm
optical pathlength optical glass cuvettes. Concentration of
AuNPs suspensions were determined by the absorbance
intensity at 400 nm as described by Scarabelli et al. [
Transmission electron microscopy (TEM)
All samples were measured with an FEI Tecnai spirit
TEM (FEI, Hillsboro, Oregon, USA) at 120 kV. Images
were recorded with a Veleta CCD camera 2048 × 2048
(Olympus-SIS, Münster, Germany) or Eagle CCD camera
4096 × 4096 (FEI, Hillsboro, Oregon, USA) and processed
using ImageJ software as described in the supporting
information (Section 1 of Additional file 1: Supplementary
Single AuNPs were characterized using conventional
TEM: Briefly, single AuNPs suspension (10 μL) was
deposited onto a 400 mesh carbon-coated copper grid and
let dried at room temperature. Images were recorded
with the Veleta camera.
Aggregated AuNPs were characterized using cryo-TEM:
Aggregated AuNPs suspension (5 μL) was deposited on a
carbon-coated copper grid and liquid excess was carefully
removed with filter paper. The grid was then plunged into
a liquid ethane bath cooled by liquid nitrogen. The
resulting vitrified sample was then stored in liquid nitrogen prior
to analysis. Images were recorded using the Eagle camera.
Depolarized dynamic light scattering (DDLS)
Light scattering data were collected at constant
temperature (21 °C) at θ = 15°, using a commercial goniometer
instrument (3D LS Spectrometer, LS Instruments AG,
Switzerland). The primary beam was formed by a linearly
polarized and collimated laser beam (Cobalt 05–01 diode
pumped solid state laser, λ = 660 nm, P max. = 500 mW),
and the scattered light was collected by single-mode optical
fibres equipped with integrated collimation optics. The
collected light was coupled into two high-sensitivity APD
detectors via laser-line filters (Perkin Elmer, Single Photon
Counting Module), and their outputs were fed into a
twochannel multiple-tau correlator. The signal-to-noise ratio
was improved by cross-correlating these two channels.
With respect to the primary beam, depolarized scattering
was observed via cross-polarizers. The incoming laser beam
passed through a Glan-Thompson polarizer with an
extinction ratio of 10−6, and another Glan-Thompson polarizer,
with an extinction ratio of 10−8, was mounted in front of
the collection optics. To estimate the number-averaged
particle size distribution, the DDLS spectra were analyzed by
the approach presented elsewhere [
3D human epithelial tissue barrier model
Experiments were carried out using the human alveolar
epithelial type II cell line A549 [
], human blood
monocyte-derived macrophages (MDM) and dendritic cells
(MDDC). A549 cell line was obtained from the American
Type Culture Collection (ATCC, USA), while human
whole blood monocytes (MDM and MDDC) were isolated
from buffy coats provided by the blood donation service
SRK Bern and purified using CD14 Microbeads (Milteny
]. Cells were maintained in RPMI 1640 (Gibco,
Life Technologies Europe B.V., Zug, Switzerland)
supplemented with 10% (v/v) fetal bovine serum (FBS; PAA
Laboratories, Chemie Brunschwig AG, Basel, Switzerland),
1% (v/v) L-Glutamine (Life Technologies Europe) and 1%
(v/v) penicillin/streptomycin (Gibco) and placed in a
humidified incubator (37 °C, 5% CO2). A549 cells
were subcultured through trypsinization when reached
near-confluence and medium was changed every 3 days.
Initial cell concentrations were calculated using trypan
blue exclusion method (0.4% Trypan blue solution, T8154;
Sigma Aldrich). The working cell concentrations were
prepared by diluting cells with cell culture medium.
3D co-culture model
The co-cultures were prepared as previously described
]. Briefly, A549 cells (54⋅104 cells/mL, 0.5 mL, upper
chamber) were seeded on a transparent BD Falcon cell
culture inserts (surface area of 0.9 cm2, pores of 3.0 μm
diameter, PET membranes for 12-well plates; BD Biosciences)
placed in a BD Falcon tissue culture plates (12-er well
plates; BD Biosciences) containing 1.5 mL medium (lower
chamber). Cells were cultured for 4 days and the medium
was changed after the 2nd day. On day 5, medium was
removed from the upper and lower chambers, the inserts
were turned up-side down, placed in a petri dish and cells
at the bottom of the membrane were gently abraded with a
cell scraper. MDDCs (84⋅104 cells/mL, 65 μL) were then
pipetted onto the bottom side of the inserts and incubated
for 70 min. Afterwards, the insert were placed back into
the well plate containing 1.5 mL fresh medium. Finally
MDMs (2.5⋅104 cells/mL, 0.5 mL) were added on top of
the A549 cells prior to be incubated for another 24 h. The
cells were then transferred from submerged to air-liquid
interface conditions 24 h prior to be exposed. On day 6,
medium was removed from the upper chamber and
medium form the lower chamber was replaced with
0.6 mL of fresh medium.
Characterization of co-culture model with laser scanning microscopy (LSM)
LSM description: Samples were acquired using LSM 710
Meta with an inverted Zeiss microscope (Carl Zeiss
GmbH, Jena, Germany). The z-stack images of the cells
were acquired using 20× and 40× magnification lens with
numerical aperture with numerical aperture, NA 0.75 and
1.3, respectively. Image processing was performed using
ImageJ software and 3D rendering with Imaris software.
Visualization of the 3D co-culture model: In order to
obtain a clear characterization of the 3D co-culture model,
each cell type was stained with different fluorophores
(Vybrant® multicolor cell labeling kit, Invitrogen
Molecular Probes) prior to the co-culture composition. Briefly,
MDDCs and MDMs (1.106 cells/mL in RPMI 1640) were
treated with vibrant dye DiI and DiD, respectively, (5 μL
for 1 mL of cell suspension) and incubated for 20 min in
the incubator. Cells were centrifuged and washed with
RPMI 1640 3 times prior to be seeded. In the meantime,
the layer of A549 grown on the insert was treated with
vibrant dye DiO (1.5 μL in 200 μL of RPMI 1640) and
incubated for 20 min in the incubator. Cells were washed
twice with RPMI 1640 for 10 min. The co-cultures were
then composed as previously described except that MDMs
were seeded at a higher density (4⋅104 cells/mL) in order
to improve their visualization in the window frame.
Air-liquid Interface cell exposure
The cells were exposed to AuNPs at the air-liquid
interface using the Vitrocell® Cloud exposure system. It
consists of three main parts: a nebulizer, an aerosol chamber
and a base module constituted of 12-well size inserts and
connected to a controlled heating unit. The aerosol is
generated into the exposure chamber via a vibrating mesh
(Aeroneb®Pro, Aerogen, with a span of 2.5–6.0 μm volume
mean diameter). The Vitrocell® Cloud exposure system
allows for a dose-controlled and uniform deposition.
Cells were exposed at the air-liquid interface to single and
aggregated AuNPs by nebulization of 200 μL of AuNPs
suspension in 0.5 mM NaClaq at the specific concentrations
of 0.05, 0.10, 0.25 and 0.50 mg/mL (concentrations
determined by UV-Vis measurements). Exposure to 200 μL of
0.5 mM NaClaq only was done as negative control. After
10 min exposure, deposition of cloud was complete and
the TCCC were kept at the air-liquid interface in fresh
medium for post-exposure incubation times of 4, 24 and
48 h in the incubator.
The deposition of AuNPs after nebulization was analyzed
by inductively coupled plasma optical emission
spectroscopy (ICP-OES) and transmission electron microscopy
(TEM) to determine the mass of gold and the number of
particles (i.e. single or aggregates), respectively.
For each exposure condition (single/aggregated AuNP
and concentration), a single slot copper grid was
exposed to nebulized AuNPs. Deposition was repeated in
triplicate for each condition. Automatic acquisition of 25
images per grid was recorded using the Eagle camera at
a magnification of 18,500X (image size 2.39 × 2.39 μm).
Images containing obvious large artefacts (i.e. dirt, grid
edge) that interfered with the automated thresholding
were excluded prior to the image processing, since these
data yield biased results. Also empty pictures were not
processed. Image processing was performed on a stack
of images as described in the supporting information
(Section 1 of Additional file 1: Supplementary information)
and the number of analyzed particles is also reported. The
output of the analysis is the number of events (single
particles or aggregates) and their associated area. Hence, the
number deposition (single and aggregated AuNPs/cm )
and surface deposition (% area) can be obtained by the
ratio of these results and the total surface area of all
pictures (including empty pictures).
Mass deposition was determined by addition of the mass
of each fraction (apical, intracellular and basal). See
Cell morphology, cell layer integrity, cytotoxicity and
(pro)-inflammation of co-culture model exposed to
AuNPs were evaluated and compared to co-culture model
exposed to 0.5 mM aqueous NaCl as negative control.
Assays were repeated in 3 individual experiments.
Cell morphology after exposure was analyzed by LSM (see LSM description above)
After particle exposure and post-incubation, the samples
were washed with PBS, fixed with paraformaldehyde (4% in
PBS, Sigma Aldrich) for 10 to 15 min and washed twice
with PBS. The samples were incubated with DAPI
(4′,6diamidino-2-phenylindole, Sigma Aldrich) (1:100, nuclei
stain) and rhodamine phalloidin (1:50, F-actin cytoskeleton
stain, Life Technologies) in Triton X-100 solution (0.2% in
PBS, to permeabilize the cell membrane, Sigma Aldrich) for
60 min. Afterwards, the samples were washed with PBS
(3 times) prior to be mounted on objective slides in
glycergel mounting medium (Dako).
Cell layer integrity
Permeability to fluorescein isothiocyanate (FITC) coupled
to dextran 70 kDa (Sigma Aldrich) was used to assess cell
layer integrity. Intact epithelial cell layer grown on a
membrane forming tight junction should prevent paracellular
transport, and so transport of FITC dextran applied on the
apical side is low. Briefly, after post-exposure time, TCCC
was rinsed with RPMI without phenol red and the medium
in the basal side was replaced by 1.5 mL of RPMI without
phenol red. At the apical side, 500 μL of 1 mg/mL FITC
dextran in RPMI without phenol red was applied and
incubated for 1 h. As a positive control, EDTA
(Ethylenediaminetetraacetic Acid, Sigma Aldrich) 10 mM
in RPMI without phenol red was added to the FITC dextran
solution applied at the apical side. Afterwards, medium in
the lower side was collected and the passage of FITC
dextran was quantified by measuring fluorescence of
the sample in triplicate using a Multireader microplate
(λex/λem = 490/520 nm). The fluorescence was normalized
to the translocation of the FITC dextran trough an empty
The cytotoxicity was assessed by measuring the release
of the cytosolic enzyme lactate dehydrogenase (LDH)
into the medium (basal compartment) that is indicative
of cell membrane damage. LDH was quantified using the
LDH cytotoxicity detection kit (Roche Applied Science,
Germany) according to the manufacturer’s guidelines and
absorbance was read at 490 nm using a microplate reader
(Bio-Rad). The sample was measured in triplicate, sample
absorbance was corrected by subtracting medium
absorbance and values were then normalized to the negative
control. As a positive control, TCCC were treated with
100 μL of 0.2% triton X100 in PBS at the apical side and
placed in the incubator for the post-exposure time.
Positive control sample was diluted 10 times prior to be
measured to remain in the linear part of the LDH activity and
the absorbance was then multiply by the diluting factor to
express the normalized LDH activity.
The (pro)-inflammation response was investigated by
quantifying the tumor necrosis factor α (TNF-α) release
into the medium (basal compartment) using the DuoSet
ELISA Development Kit (R&D Systems) according to
the manufacturer’s protocol and absorbance was read at
450 nm using a microplate reader (Bio-Rad). The sample
was measured in triplicates, sample absorbance was
corrected by subtracting medium absorbance. As a positive
control, TCCC were treated with 600 μL of LPS 1 μg/mL
in medium at the basal side and placed in the incubator
for the post-exposure time.
For each exposure condition (i.e. AuNPs type,
concentration and time), apical, intracellular and basal fractions
were collected from 3 different inserts and the amount
of gold was determined using ICP-OES. Each exposure
condition was repeated in 4 individual experiments.
First, the basolateral medium was collected. Then, to
recover particles deposited on the apical side that have not
been associated with cells, the cell layer was washed
twice with PBS (300 μL). Finally, the cells were collected
by scrapping the cells after treatment with
trypsinEDTA (600 μL) for approximatively 25 min at 37 °C.
The obtained samples were stored at −80 °C until
Microwave-assisted acidic digestion of AuNP samples
was conducted in the microwave Multiwave Pro (Anton
Paar, Germany) which is equipped with two standard
magnetrons of 850 W able to deliver a microwave power
up to 1500 W in an unpulsed mode over the full power
range. The applied microwave energy is controlled by
contactless sensors for internal temperature and vessel
pressure and by IR sensor which is equipped with a
temperature sensor, preventing overheating, and an IR
sensor monitors the temperature of vessels. Rotor
24HVT50 was used with pressure vessels HVT50 made
of PTFE-TFM. Briefly, samples were gradually defrosted
from −80 °C → -20 °C → 4 °C → room temperature.
Samples were transferred into vessels and were treated
with HNO3 70% (0.6 mL, Sigma Adrich) and H2O2
30% (0.3 mL, Merck). The volume was completed to
3 mL with millipore H2O (0.3 mL). Samples were placed
in the microwave and the mixture was irradiated at
100 °C (ramp for 10 min, hold 10 min) and further at
140 °C (ramp for 10 min, hold for 10 min) and finally
cooled to 70 °C (ramp 12 min) with a maximum power of
600 W. Samples was then transferred into 15 mL falcon
tubes, and each vessel was rinsed with millipore H2O
(0.5 mL). Finally samples were completed with millipore
H2O, if necessary, to a final volume of 3.5 mL.
ICP-OES analyses were carried out on an Optima 7000
DV, Perkin Elmer. Measurements were performed at a
wavelength of 242.8 nm, at an axial plasma view. The
plasma flow was 15 L/min and the sample flow rate
1.5 mL/min. Calibration was performed using a gold
standard for ICP (1001 mg/L ± 2 mg/L, Fluka) for each
matrix from 0.025 to 0.5 μg/mL. Samples were measured
in triplicate and a washing step was performed by the
instrument between each exposure condition. Analysis was
repeated 4 times for each exposure condition. The
obtained concentration (μg/mL) was multiplied by the
sample volume (3.5 mL) to determine the Au mass.
Particle localization in the co-culture by TEM
Sample preparation: All chemicals were obtained from
Polysciences, unless otherwise stated. The exposed cells
on the transwell membrane were fixed with
glutaraldehyde 2.5% in HEPES buffer (0.03 M) for at least 48 h at
4 °C. Then, they were washed three times at room
temperature with sodium cacodylate buffer (0.1 M) under
gentle stirring for at least 5 min per washing step. The
samples were then post-fixed with OsO4 (1% in 0.1 M
sodium cacodylate buffer) for 2 h and washed again three
times with sodium cacodylate buffer as described before.
The dehydration of the samples was achieved using a
series of gradually increasing ethanol concentration
(30, 50, 70, 80, 96 and 100% dried over molecular sieve),
each step lasting 15 min followed by gradual increasing
concentrations of the epoxy embedding polymer in 100%
ethanol dried over molecular sieve (30% for 1 h at 4 °C,
70% overnight at 4 °C, 100% for 2 h at room temperature,
repeated twice). The samples were then polymerized at
60 °C for 72 h. From the polymerized epoxy resin blocks,
ultrathin sections of 80 nm (gray-sliver reflection) were
sectioned perpendicular to the Transwell membrane by a
Ultra 35° diamond knife (Diatome, Nidau, Switzerland)
using a Leica Ultracut UC6 ultramicrotome (Leica
Microsystems, Wetzlar, Germany). These sections were
then brought onto Formvar film coated single slot copper
grids (PlanoEM, Wetzlar, Germany) and stained with
uranyl acetate and lead citrate in a Leica EM Stain citrate
(Leica Microsystems, Wetzlar, Germany). TEM images
were recorded using the Veleta camera, with a resolution
of 11.2 nm/pixel (overviews) and 0.78 nm/pixel (details).
Number of repetitions is given in the respective
paragraph. Individual experiments mean different
aerosolization and cell culture with different passage number and
different monocytes isolations. Data are presented either
as mean ± standard deviation or mean with data points.
Statistics were run using ORIGINLAB. Statistical
differences were determined by comparison of the means
using one-way ANOVA and Tukey’s honest significance
difference test (p = 0.05).
Particle AuNPs synthesis and characterization
Single and aggregated AuNPs were prepared adapting
the method developed by Hirsch et al. (Fig. 1a) [
Citrate-capped AuNPs were covalently functionalized
with tiopronin forming AuNPs which then displayed
carboxylic groups on the surface. As shown in the previous
study, protonation of these carboxylic groups allows the
formation of controlled aggregates. The agglomeration
process is driven by electrostatic attraction between two
tiopronin-AuNPs resulting from the hydrogen bonds
formation, hence replacing the electrostatic repulsion
when carboxylic groups are negatively charged.
Agglomeration was then stopped by addition of a polymer
mixture composed of polyvinyl alcohol and polyallyl amine
(PVA/PAAm). PVA/PAAm is important for stabilizing
the agglomerates to aggregates, because (i) amine groups
act as proton sponge, (ii) they interact with the negative
charges of the tiopronin-AuNPs and (iii) the overall
PVA/PAAm coating prevents the reversal of the
selfassembly at physiological pH. Single tiopronin-AuNPs
were also coated with PVA/PAAm polymers to have
similar chemical surface properties.
The synthesized single and aggregated AuNPs were
then characterized by UV-Vis spectroscopy, TEM, DDLS
and zeta-potential, the results are summarized in the
table (Fig. 1b). UV-Vis spectra (Fig. 1c) showed a red
shift from 524 to 574 nm and a broadened localized
surface plasmon resonance (LSPR) band for the aggregates
in comparison to the single NPs showing the formation
of the aggregates. TEM images and DDLS
measurements further confirmed the size and state of dispersion
of the different samples. Single AuNPs showed a core
size of 14.5 nm (TEM) (Fig. 1d, Additional file 1: Figure S5)
and a hydrodynamic diameter of 32 ± 12 nm (DDLS). The
difference of size given by TEM and DDLS was due to the
large PVA polymer coating. TEM images of aggregated
AuNPs were taken at cryogenic temperature to prevent
aggregates resulting from drying artefacts (Fig. 1e,
Additional file 1: Figure S5). TEM images analysis showed
aggregates with heterogeneous population and a mean of 4
single AuNPs per aggregate (Additional file 1: Figure S6).
DDLS measurements confirmed also the aggregates
formation giving a hydrodynamic diameter of 106 ± 32 nm.
Finally, the near neutral or positive zeta potential of single
and aggregated AuNPs indicates the presence of the PVA/
PAAm coating (Additional file 1: Table S2).
AuNP aerosolization and characterization of deposition
Deposition of the aerosolized single and aggregated
AuNPs was thoroughly analyzed using ICP-OES and
TEM images obtained from the deposited single
and aggregated AuNPs showed that the particle state
(i.e. single and aggregate) was generally not affected by
aerosolization and deposition processes (Fig. 2a). The
particle diameter remained similar to prior aerosolization.
Deposition was quantified on a specific surface area by
determining (i) the mass of deposited gold using ICP-OES,
(ii) the number and (iii) the surface of deposited single or
aggregated AuNPs using TEM, resulting in the mass
position (ng/cm ), the number deposition (# events/cm )
and the surface deposition (%), respectively. The results are
summarized in the table as mean values for each
aerosolized concentration of single and aggregated AuNPs
(Fig. 2b). TEM images analysis processing is explained in
the supplementary information (Section 1 of Additional
file 1: Supplementary information) and a 3D
reconstruction of deposited aggregated is shown in Fig. 2c. Analysis
of the mass deposition expressed in function of the
aerosolized dose (mg/mL), showed that the deposition
was reproducible and proportional to the applied dose for
both AuNPs systems (Fig. 2d, linear fit r2 > 0.99). It is
important to note that the deposited mass obtained after
aerosolization of single and aggregated AuNPs were not
statistically different and were simplified to an
approximate mass, from 30 to 300 ng/cm2, for further discussions
(Fig. 2b). On the other hand, deposition quantification
counting the number of events, showed an important
difference between single and aggregated AuNPs,
especially at the highest doses (Fig. 2e). Numbers of single
AuNPs were 10 to 30 fold more than the aggregated ones
at the deposited mass of 150 and 300 ng/cm2, respectively.
Interestingly, while the number of deposited single AuNPs
increased proportionally to the deposited mass, the
number of deposited aggregates did not increase at doses
above 150 ng/cm2. Indeed, visualization of the TEM
images showed that at the highest concentrations (Fig. 2a,
0.50 mg/mL), aggregates tended to form bigger clusters
while single particles remained mainly dispersed. Finally,
analysis of the surface deposition showed that the
deposited single and aggregated NPs covered similar total surface
area which increased proportionally with the deposited
mass (Fig. 2f ). This is also supporting the previous
observation of a clustering effect with the aggregates at higher
concentration. Indeed, to obtain the same total surface area
as the single NPs, the individual deposited aggregated
NPs must have larger surface area to compensate the
lower number deposition. Determination of the number
of particles per aggregates showed an increase at the
highest concentration (300 ng/cm2) (Additional file 1:
Figure S6). The larger size of the deposited aggregates at
higher concentration could be the result of
agglomeration occurring during nebulization, as also observed for
higher concentration of nebulized superparamagnetic
iron oxide NPs [
] and/or the result of the drying
artifact of the more concentrated nebulized droplets.
Characterization of the 3D human epithelial tissue barrier after AuNPs exposure
The in vitro lung model was first characterized by LSM.
As depicted in Fig. 3a, the three cell types, each
represented in a different color, were visualized. The presence
of the apical layer composed of a monolayer of alveolar
cells (green) with incorporated MDMs (red), while at the
basal side MDDCs (orange) were observed.
No noticeable change was observed in the cell
morphology after exposure to different concentrations of single
and aggregated AuNPs in comparison to control cultures
exposed to NaClaq solution only (Fig. 3b-d).
The epithelial integrity of the cell layer after exposure
was also assessed by testing the FITC-dextran (70 kDa)
permeability. As shown in Fig. 3e, FITC-dextran
translocation across the barrier 24 h after exposure to
NaClaq solution was very low (1.95%) and remained low
after 48 h (1.80%). Similar translocation rates were found
for cells exposed to the highest concentration of single
and aggregated AuNPs. In contrast, when the cells were
treated with EDTA, a chelator agent known to open
the tight junctions, dextran translocation increased to
Cell response after AuNPs exposure
Post-exposure cytotoxicity was assessed by measuring
the release of lactate dehydrogenase enzyme into the
medium. As shown in Fig. 4, cell exposure to single and
aggregated AuNPs up to the highest dose of 300 ng/cm ,
did not show any apparent membrane damage 4, 24 and
48 h in comparison to saline solution.
(Pro)-inflammation response to AuNPs exposure was
assessed by measuring the released cytokine TNFα. No
TNFα release has been measured in comparison to the
positive control indicating that the particles did not
induce any pro-inflammatory reactions (data not shown).
Biodistribution behavior of AuNPs and localization in the cells
After exposure onto the lung cell surface, AuNPs can
either remain on the apical surface, being taken up by
cells and/or translocated across the cell layer into the
basolateral compartment. The AuNPs concentration was
determined by ICP-OES for three different
compartments: (i) air-exposed (apical) cell side to measure the
AuNPs deposited on the apical cell surface, (ii) the
intracellular AuNPs content, and (iii) the medium in the
lower (basal) compartment to determine the
translocated particles. The cultures were first exposed to single
and aggregated AuNPs at different concentrations, i.e.
30, 60, 150 and 300 ng/cm2, and the gold distribution
was analyzed 24 h after exposure (Fig. 5a). For both
particle types and all concentrations less than 5% gold was
found in the apical fraction and the majority of gold, i.e.
more than 90% of the total applied gold mass, was found
in the intracellular fraction (Additional file 1: Figure S7
shows amount of gold found intracellularly). Only
exposure to single AuNPs at the lowest concentration of
30 ng/cm2 was slightly different as 12% of gold was
found in the apical fraction and 85% intracellularly.
However, for all conditions only a minor fraction of gold
was found in the basolateral compartment showing
minor translocation rates between 2 and 5%. Thus, the
majority of single or aggregated AuNPs were taken up
by the cells. Moreover, the translocation rate did not
increase with increasing concentration.
Then, the biodistribution, i.e. uptake and translocation
across the barrier, of exposed single and aggregated
AuNPs was assessed at different time points, i.e. 4, 24
and 48 h at a dose of 300 ng/cm2 (Fig. 5b and c). For
single AuNPs the cellular fraction after 4 h was 84%,
increased to 94% after 24 h, and remained constant
afterwards. For aggregated AuNPs 94% of the deposited
mass was detected intracellularly after 4 h and remained
constant. The results showed that the aggregated AuNPs
were taken up faster than single AuNPs (94 vs 84%,
respectively, at 4 h post-exposure), however no difference
could be observed for the translocation rate.
Ultrathin sections of cells exposed to AuNPs were
visualized using TEM to observe the cellular localization
of AuNPs (Fig. 6). After 24 h the majority of single and
aggregated AuNPs were found intracellularly. Some of
the particles were found to be attached to the outer
apical cell surface and no particles were observed in the
intercellular space. The AuNPs were found in all three
cell types, i.e. MDM and epithelial cells on the upper
insert surface, and even to a minor extent in MDDC on
the basal side of the insert. Most of the AuNPs were
localized in vesicles and only rarely in the cytoplasm.
Attached or internalized aggregated AuNPs resulted in
spot containing much higher density of AuNPs which is
in line with the deposition characterization.
Lung is the first portal of entry into our body to airborne
particles, which have been associated to lung and
cardiovascular diseases [
]. Aggregated NPs are a major
form of airborne particles [
]. Their low effective
density compared to single particles of similar size increases
their mobility and allow them to penetrate and deposit in
the deep lung region [
]. However their behavior at the
lung barrier is poorly studied, therefore gaining a better
understanding of the aggregates interaction and fate at the
human alveolar epithelial tissue barrier is important. In
this study, an approach combining air liquid interface and
advanced lung cell co-culture has been used representing
a more realistic perspective when compared to submerged
]. Although the system has its limitation, i.e.
it is not possible to follow the long-term fate of the
particles and/or drugs in the blood as well as lymph
circulations and secondary organs, it has been shown to give
comparable results to in vivo data for short-term
translocation kinetics, i.e. up to 24 h, of apically applied
] or drugs [
AuNPs were used as model particles to study the
interaction and biodistribution of single and aggregated NPs
with lung cells after aerosol deposition. The well-controlled
chemistry to synthesize AuNPs allowed the preparation of
well-defined and stable aggregates composed of primary
AuNPs with a core diameter of 14.5 nm, and a
hydrodynamic diameter of 106 nm. These aggregated AuNPs
were compared with their corresponding single AuNPs
with a hydrodynamic diameter of 32 nm. Moreover, the
scattering and absorption properties due to the LSPR are
important aspects for their characterization and, together
with possibility of gold traces quantification, they allow for
exact characterizations including quantification [
Deposition of single and aggregated AuNPs after
aerosolization using the air liquid interface cell exposure
Cloud system was thoroughly characterized using TEM
and ICP-OES techniques expressing deposition
concentration in mass, number of entities and recovered surface
area per surface area. Dose-controlled and reproducibility
of the deposition were confirmed by the two techniques.
At the highest concentration of 150 and 300 ng/cm ,
aggregated NPs greatly reduced the number of deposited
AuNP entities of 10 to 30 fold, respectively, which may
have an impact on further cellular response. Furthermore,
these analyses allowed to appreciate the deposition at a
cellular level, as epithelial cells (A549) have a diameter of
about 14 μm as reported by Jiang et al. [
], giving a total
cellular surface of around 153 μm2, there is evidence to
assume that even at lowest dose exposure of 30 ng/cm2,
each cell should theoretically be in contact with 66
particles (Fig. 2b, applied dose: 0.05 mg/mL, deposition:
30 ng/cm2, 0.43 particles/μm2).
Exposure to single and aggregated AuNPs did not
induce any significant adverse cellular effect regarding
cytotoxicity, epithelial cell layer integrity and
(pro-)inflammation. These results are in agreement with other
studies who showed the biocompatible properties of
AuNPs in vitro or in vivo [
]. The deposited mass of
gold was in the range of 150 and 300 ng/cm2 which is
higher as what is reported in animal studies, e.g. 0.2 to
8 ng/cm2 [
] or 3.3 ng/cm2 [
]. However, since there
is no clear answer about the physiological relevance, i.e.
occupation or biomedical concentration of gold
nanoparticles via inhalation, and the aim was to compare the
translocation rate of single AuNPs vs. aggregates the
conditions producing reproducible deposition values
without inducing any cytotoxicity were chosen.
The biodistribution of the AuNPs in the lung cells
24 h after exposure showed that majority of both single
and aggregated particles were taken up and retained
inside the cells, only a minor fraction translocated across
the epithelial tissue layers, i.e. between 1.3 and 4.5%.
The translocated fraction observed in this study is in
agreement with another in vitro study where aerosolized
18-nm citrate AuNPs were exposed to a A549 epithelial
cell monolayer where a translocation rate of 2% had been
]. In vivo studies also reported only minor
translocation of AuNPs across lung in rats, 0.5% after
instillation of sulfonated triphenylphosphine AuNPs (18 and
200 nm) [
] and 1.4% after inhalation of citrate AuNPs
agglomerates (peak diameter 45 nm) [
ICP-OES measurements showed that the uptake and/
or translocation are fast processes as majority of, if not
all, AuNPs were taken up and translocated already 4 h
after exposure. This is in agreement with in vivo studies
of inhaled AuNPs: one study in mice concluded that
AuNPs (21 nm) were translocated after a short time,
inferior to 2 h [
] and another study in rats investigating
translocation over time showed that translocation of
AuNPs (18–80 nm) was complete after 1 h [
The rapid translocation and the similar low rate
(1.3–4.5%) regardless of the different deposited
concentrations let suggest that translocation of single and
aggregated AuNPs occurs through an active transcellular
transport (or transcytosis) [
]. This hypothesis is further
supported with TEM observations: 1) no AuNPs were
found in the intercellular space; 2) most of the
intracellular AuNPs were found in vesicles; 3) presence of AuNPs
in MDDCs on the basal side of the membrane. Similar
observations were found in stereological analysis of mice
lung tissue after exposure to 21-nm AuNPs [
Although, aggregated AuNPs behavior was similar to
single AuNPs regarding distribution, the only difference
found was that aggregated AuNPs were faster observed
intracellularly in comparison to single AuNPs (Fig. 5b).
Indeed, it is well known that particle size and shape
influence cellular uptake [
]. However, this
observation can be surprising since preferential uptake is
commonly expected with particles of the size around 50 nm
and with spherical shape . The faster uptake of
aggregated AuNPs could be explained by i) the larger
surface area of the aggregated AuNPs; ii) a different cellular
uptake pathway; iii) the lower number of deposited
particles for aggregates. Firstly, several findings support the
theory that larger surface area in contact with cell
membrane allow for more multivalent ionic interactions
explaining a faster or higher NPs uptake. For instance, a
study investigating internalization of NPs with various
sizes and shapes, found that in cylindrical NPs of similar
volume, particles with higher aspect ratio were
internalized faster, suggesting a favored internalization for the
larger surface area [
]. Similar observations were found
in a study comparing cellular uptake of single and
aggregated transferrin coated AuNPs (30 and 98 nm, respectively)
in which aggregated AuNPs uptake were 2-fold higher in
comparison to single AuNPs in cells expressing few
transferrin receptors [
]. Secondly, the difference observed in
uptake kinetics could also be explained by a different
uptake mechanism. Indeed, Kreyling et al. have observed a
different translocation behavior with the bigger 200 nm
AuNPs in comparison to 18–80 nm AuNPs, suggesting
that these two NPs categories were endocytosed and/or
exocytosed via different pathway [
]. Furthermore, a
study investigating shape effect of mesoporous silica NPs
found that spherical NPs were preferentially internalized
via clathrin-mediated pathway while higher aspect ratio
NPs favored caveolae-mediated pathways [
]. Finally, as
shown in Fig. 2f, at a deposition of 300 ng/cm , the
number of deposited aggregated NPs is 30 times less than
for single AuNPs.
In this study, the biodistribution of aerosolized single
and aggregated AuNPs was investigated using a 3D
model of the human epithelial tissue barrier. Robust
characterization was used to evaluate the exact delivered
dose onto the cell surface and to determine the cellular
uptake and translocation across the barrier. Overall, we
found that within a short time (<4 h), the majority of the
AuNPs, single or aggregated, were taken up and retained
inside the cells while only a minor fraction translocated
to the basal side (<5%). The low translocation rate is
similar to the ones found for AuNPs in vivo highlighting
the possibility of using a sophisticated in vitro approach
to predict in vivo biokinetics of inhaled AuNPs. Finally,
at higher concentration (300 ng/cm ) the aggregated
AuNPs showed a significant reduction of the number of
deposited spots and a faster cellular uptake but only
during the first time points assessed, however, no significant
change of the translocation rate was observed. Hence,
aggregation is fundamental for the cellular uptake
kinetics of NPs during the first hours after exposure and has
to be considered, either in a biomedical setting of drug
delivery or for hazard assessment.
Additional file 1: Supplementary information. (PDF 765 kb)
We thank P Lemal and D Urban for their help in performing cryo-TEM and
ICP-OES measurements, respectively.
This study was supported by the Toyota Motor Corporation and grants of
the Swiss National Science Foundation (Grant # 310030_159847 / 1) and the
work benefitted from support from the Swiss National Science Foundation
through the National Centre of Competence in Research Bio-Inspired
Materials, and the Adolphe Merkle Foundation.
Availability of data and materials
The datasets generated and/or analyzed during the current study are not
publicly available due to their large storage space (> 100 Gigabyte) but are
available by FTP link from the corresponding author on reasonable request.
ED participated in the design of the study, carried out all chemical synthesis,
biological based experimentation and drafted the manuscript. DV was involved
in the TEM analysis and characterization of the AuNPs, LR-L supported the
particle synthesis and characterization, FD supported air-liquid interface cell
exposure and biological experiments, SB was involved in the DLS measurement
and analysis, DS and JB were involved in performing LSM imaging. AP-F and
BR-R were involved in the planning and technical advisory of the study. BR-R
was the project leader; she was involved in the planning the design of the
study, has intellectually accompanied all experimental work, made substantial
contributions to the analysis and interpretation of the data. BR-R, LR-L and
DV have been involved in critically revising the manuscript for important
intellectual content. All authors have read and approved the final manuscript.
Ethics approval and consent to participate
The need for ethics approval or consent to participate is not applicable.
Consent for publication
The need for consent for publication is not applicable.
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
1. Moore TL , Rodriguez-Lorenzo L , Hirsch V , Balog S , Urban D , Jud C , et al. Nanoparticle colloidal stability in cell culture media and impact on cellular interactions . Chem Soc Rev . 2015 ; 44 : 6487 - 305 . doi: 10 .1039/c4cs00487f.
2. Stark WJ . Nanoparticles in biological systems . Angew Chemie - Int Ed. 2011 ; 50 : 1242 - 58 .
3. Wick P , Manser P , Limbach LK , Dettlaff-Weglikowska U , Krumeich F , Roth S , et al. The degree and kind of agglomeration affect carbon nanotube cytotoxicity . Toxicol Lett . 2007 ; 168 : 121 - 31 .
4. Nichols G , Byard S , Bloxham MJ , Botterill J , Dawson NJ , Dennis A , et al. A review of the terms agglomerate and aggregate with a recommendation for nomenclature used in powder and particle characterization . J Pharm Sci . 2002 ; 91 : 2103 - 9 .
5. Puri R , Richardson TF , Santoro RJ , Dobbins RA . Aerosol dynamic processes of soot aggregates in a laminar ethene diffusion flame . Combust Flame . 1993 ; 92 : 320 - 33 .
6. Maricq MM , Xu N. The effective density and fractal dimension of soot particles from premixed flames and motor vehicle exhaust . J Aerosol Sci . 2004 ; 35 : 1251 - 74 .
7. Van Gulijk C , Marijnissen JCM , Makkee M , Moulijn JA , Schmidt-Ott A . Measuring diesel soot with a scanning mobility particle sizer and an electrical low-pressure impactor: performance assessment with a model for fractal-like agglomerates . J Aerosol Sci . 2004 ; 35 : 633 - 55 .
8. Rissler J , Swietlicki E , Bengtsson A , Boman C , Pagels J , Sandström T , et al. Experimental determination of deposition of diesel exhaust particles in the human respiratory tract . J Aerosol Sci . 2012 ; 48 : 18 - 33 .
9. Steiner S , Bisig C , Petri-Fink A , Rothen-Rutishauser B . Diesel exhaust: current knowledge of adverse effects and underlying cellular mechanisms . Arch Toxicol . 2016 . doi: 10 .1007/s00204-016-1736-5.
10. Van Setten BAAL , Makkee M , Moulijn JA . Science and technology of catalytic diesel particulate filters . doi:101081/CR-120001810 . 2007 .
11. Zhu J , Lee KO , Yozgatligil A , Choi MY . Effects of engine operating conditions on morphology, microstructure, and fractal geometry of light-duty diesel engine particulates . Proc Combust Inst . 2005 ; 30 : 2781 - 9 . doi: 10 .1016/ j.proci. 2004 . 08 .232.
12. Rissler J , Messing ME , Malik AI , Nilsson PT , Nordin EZ , Bohgard M , et al. Effective density characterization of soot agglomerates from various sources and comparison to aggregation theory . Aerosol Sci Technol . 2013 ; 47 : 792 - 805 . doi: 10 .1080/02786826. 2013 . 791381 .
13. Burtscher H . Physical characterization of particulate emissions from diesel engines: a review . J Aerosol Sci . 2005 ; 36 : 896 - 932 .
14. Heyder J , Gebhart J , Rudolf G , Schiller CF , Stahlhofen W. Deposition of particles in the human respiratory tract in the size range 0 . 005 - 15 μm. J Aerosol Sci . 1986 ; 17 : 811 - 25 .
15. Oberdörster G , Oberdörster E , Oberdörster J . Nanotoxicology: an emerging discipline evolving from studies of ultrafine particles . Environ Health Perspect . 2005 ; 113 : 823 - 39 .
16. Choi HS , Ashitate Y , Lee JH , Kim SH , Matsui A , Insin N , et al. Rapid translocation of nanoparticles from the lung airspaces to the body . Nat Biotechnol . 2010 ; 28 : 1300 - 3 . 10 .1038/nbt.1696.
17. Semmler-Behnke M , Kreyling WG , Lipka J , Fertsch S , Wenk A , Takenaka S , et al. Biodistribution of 1 .4- and 18-nm gold particles in rats. Small . 2008 ; 4 : 2108 - 11 .
18. Lipka J , Semmler-Behnke M , Sperling RA , Wenk A , Takenaka S , Schleh C , et al. Biodistribution of PEG-modified gold nanoparticles following intratracheal instillation and intravenous injection . Biomaterials . 2010 ; 31 : 6574 - 81 .
19. Kreyling WG , Semmler-Behnke M , Seitz J , Scymczak W , Wenk A , Mayer P , et al. Size dependence of the translocation of inhaled iridium and carbon nanoparticle aggregates from the lung of rats to the blood and secondary target organs . Inhal Toxicol . 2009 ; 21 ( Sup 1 ): 55 - 60 . doi: 10 .1080/08958370902942517.
20. Mühlfeld C , Gehr P , Rothen-Rutishauser B . Translocation and cellular entering mechanisms of nanoparticles in the respiratory tract . Swiss Med Wkly . 2008 ; 138 : 387 - 91 .
21. Pillay V , Murugan K , Choonara YE , Kumar P , Bijukumar D , du Toit LC. Parameters and characteristics governing cellular internalization and trans-barrier trafficking of nanostructures . Int J Nanomedicine . 2015 ; 10 : 2191 - 206 . doi: 10 .2147/IJN.S75615.
22. Kreyling WG , Hirn S , Möller W , Schleh C , Wenk A , Celik G , et al. Air-blood barrier translocation of tracheally instilled gold Nanoparticles inversely depends on particle size . ACS Nano . 2014 ; 8 : 222 - 33 . doi: 10 .1021/nn403256v.
23. Kreyling WG , Semmler M , Erbe F , Mayer P , Takenaka S , Schulz H , et al. Translocation of ultrafine insoluble iridium particles from lung epithelium to extrapulmonary organs is size dependant but very low . Environment . 2002 ; 7394 : 1513 - 30 .
24. Bruinink A , Wang J , Wick P . Effect of particle agglomeration in nanotoxicology . Arch Toxicol . 2015 ; 89 : 659 - 75 . doi: 10 .1007/s00204-015-1460-6.
25. Balasubramanian SK , Poh K-W , Ong C-N , Kreyling WG , Ong W-Y , Yu LE . The effect of primary particle size on biodistribution of inhaled gold nano-agglomerates . Biomaterials . 2013 ; 34 : 5439 - 52 .
26. Blank F , Rothen-Rutishauser B , Gehr P . Dendritic cells and macrophages form a transepithelial network against foreign particulate antigens . Am J Respir Cell Mol Biol . 2007 ; 36 : 669 - 77 . doi: 10 .1165/rcmb.2006- 0234OC .
27. Hirsch V , Kinnear C , Rodriguez-Lorenzo L , Monnier CA , Rothen-Rutishauser B , Balog S , et al. In vitro dosimetry of agglomerates. Nano . 2014 ; 6 : 7325 - 31 . doi: 10 .1039/c4nr00460d.
28. Scarabelli L , Grzelczak M , Liz-Marzán LM . Tuning gold Nanorod synthesis through Prereduction with salicylic acid . Chem Mater . 2013 ; 25 : 4232 - 8 . doi: 10 .1021/cm402177b.
29. Geers C , Rodriguez-Lorenzo L , Andreas Urban D , Kinnear C , Petri-Fink A , Balog S. A new angle on dynamic depolarized light scattering: numberaveraged size distribution of nanoparticles in focus . Nano . 2016 ; 8 : 15813 - 21 . doi: 10 .1039/C6NR03386E.
30. Lieber M , Todaro G , Smith B , Szakal A , Nelson-Rees W. A continuous tumor-cell line from a human lung carcinoma with properties of type II alveolar epithelial cells . Int J Cancer . 1976 ; 17 : 62 - 70 . doi: 10 .1002/ ijc.2910170110.
31. Steiner S , Czerwinski J , Comte P , Popovicheva O , Kireeva E , Müller L , et al. Comparison of the toxicity of diesel exhaust produced by bio- and fossil diesel combustion in human lung cells in vitro . Atmos Environ . 2013 ; 81 : 380 - 8 .
32. Graczyk H , Bryan LC , Lewinski N , Suarez G , Coullerez G , Bowen P , et al. Physicochemical characterization of nebulized superparamagnetic iron oxide nanoparticles (SPIONs) . J Aerosol Med Pulm Drug Deliv . 2015 ; 28 : 43 - 51 .
33. Pope CA , Dockery DW , Schwartz J . Review of epidemiological evidence of health effects of particulate air pollution . Inhal Toxicol . 1995 ; 7 : 1 - 18 . doi: 10 .3109/08958379509014267.
34. Donaldson K , Tran L , Jimenez LA , Duffin R , Newby DE , Mills N , et al. Combustion-derived nanoparticles: a review of their toxicology following inhalation exposure . Part Fibre Toxicol . 2005 ; 2 : 10 .
35. Paur HR , Cassee FR , Teeguarden J , Fissan H , Diabate S , Aufderheide M , et al. In-vitro cell exposure studies for the assessment of nanoparticle toxicity in the lung-a dialog between aerosol science and biology . J Aerosol Sci . 2011 ; 42 : 668 - 92 .
36. Bachler G , Losert S , Umehara Y , von Goetz N , Rodriguez-Lorenzo L , Petri-Fink A , et al. Translocation of gold nanoparticles across the lung epithelial tissue barrier: combining in vitro and in silico methods to substitute in vivo experiments . Part Fibre Toxicol . 2015 ; 12 : 18 . doi: 10 .1186/ s12989-015-0090-8.
37. Schmid O , Jud C , Umehara Y , Mueller D , Bucholski A , Gruber F , et al. Biokinetics of aerosolized liposomal Ciclosporin a in human lung cells In Vitro using an air-liquid cell Interface exposure system . J Aerosol Med Pulm Drug Deliv . 2017 ;:jamp. 2016 . 1361 . doi: 10 .1089/jamp. 2016 . 1361 .
38. Liz-marzan LM . Tailoring surface Plasmons through the morphology and assembly of metal Nanoparticles . Langmuir . 2006 ; 22 : 32 - 41 .
39. Jiang R , Shen H , Piao Y-J . The morphometrical analysis on the ultrastructure of A549 cells . Romanian J Morphol Embryol . 2010 ; 51 : 663 - 7 .
40. Ghosh P , Han G , De M , Kim CK , Rotello VM . Gold nanoparticles in delivery applications . Adv Drug Deliv Rev . 2008 ; 60 : 1307 - 15 .
41. Schleh C , Holzwarth U , Hirn S , Wenk A , Simonelli F , Schäffler M , et al. Biodistribution of inhaled gold nanoparticles in mice and the influence of surfactant protein D . J Aerosol Med Pulm Drug Deliv . 2013 ; 26 : 24 - 30 . doi: 10 .1089/jamp. 2011 . 0951 .
42. Geiser M , Quaile O , Wenk A , Wigge C , Eigeldinger-Berthou S , Hirn S , et al. Cellular uptake and localization of inhaled gold nanoparticles in lungs of mice with chronic obstructive pulmonary disease . Part Fibre Toxicol . 2013 ; 10 : 19 . doi: 10 .1186/ 1743 -8977-10-19.
43. Albanese A , Tang PS , Chan WCW . The effect of Nanoparticle size, shape, and surface chemistry on biological systems . Annu Rev Biomed Eng . 2012 ; 14 : 1 - 16 . https://doi.org/10.1146/annurev-bioeng- 071811 -150124.
44. Gratton SEA , Ropp PA , Pohlhaus PD , Luft JC , Madden VJ , Napier ME , et al. The effect of particle design on cellular internalization pathways . Proc Natl Acad Sci U S A . 2008 ; 105 : 11613 - 8 .
45. Albanese A , Chan WCW . Effect of gold Nanoparticle aggregation on cell uptake and toxicity . ACS Nano . 2011 ; 5 : 5478 - 89 . doi: 10 .1021/nn2007496.
46. Hao N , Li L , Zhang Q , Huang X , Meng X , Zhang Y , et al. The shape effect of PEGylated mesoporous silica nanoparticles on cellular uptake pathway in Hela cells . Microporous Mesoporous Mater . 2012 ; 162 : 14 - 23 . doi: 10 .1016/ j.micromeso. 2012 . 05 .040.
47. Vanhecke D , Rodriguez-Lorenzo L , Kinnear C , Durantie E , Rothen-Rutishauser B , Fink AS . Assumption-free morphological quantification of single anisotropic nanoparticles and aggregates . Nano . 2017 ; 9 : 4918 - 27 . doi: 10 .1039/C6NR07884B.