Protein corona: implications for nanoparticle interactions with pulmonary cells
Konduru et al. Particle and Fibre Toxicology
Protein corona: implications for nanoparticle interactions with pulmonary cells
Nagarjun V. Konduru 0 1
Ramon M. Molina 0 1
Archana Swami 1
Flavia Damiani 1
Georgios Pyrgiotakis 0 1
Paulo Lin 1
Patrizia Andreozzi 2 3
Thomas C. Donaghey 1
Philip Demokritou 0
Silke Krol 5 6
Wolfgang Kreyling 4
Joseph D. Brain 0 1
0 Center for Nanotechnology and Nanotoxicology, Harvard T.H. Chan School of Public Health , 665 Huntington Avenue, Boston, MA 02115 , USA
1 Department of Environmental Health, Molecular and Integrative Physiological Sciences Program, Harvard T.H. Chan School of Public Health , 665 Huntington Avenue, Boston, MA 02115 , USA
2 CIC biomaGUNE Soft Matter Nanotechnology Group, Paseo de Miramón , 182, 20014 San Sebastian-Donostia, Guipuzcoa , Spain
3 IFOM , via Adamello 16, 20139 Milano , Italy
4 Institute of Epidemiology 2, Helmholtz Zentrum München - German Research Center for Environmental Health , Ingolstädter Landstraße 1, 85764 Oberschleißheim , Germany
5 I.R.C.C.S. Istituto Tumori Giovanni Paolo II , Viale O. Flacco 65, 70124 Bari , Italy
6 Fondazione I.R.C.C.S. Istituto Neurologico Carlo Besta , Via Amadeo 42, 20133 Milan , Italy
Background: We previously showed that cerium oxide (CeO2), barium sulfate (BaSO4) and zinc oxide (ZnO) nanoparticles (NPs) exhibited different lung toxicity and pulmonary clearance in rats. We hypothesize that these NPs acquire coronas with different protein compositions that may influence their clearance from the lungs. Methods: CeO2, silica-coated CeO2, BaSO4, and ZnO NPs were incubated in rat lung lining fluid in vitro. Then, gel electrophoresis followed by quantitative mass spectrometry was used to characterize the adsorbed proteins stripped from these NPs. We also measured uptake of instilled NPs by alveolar macrophages (AMs) in rat lungs using electron microscopy. Finally, we tested whether coating of gold NPs with albumin would alter their lung clearance in rats. Results: We found that the amounts of nine proteins in the coronas formed on the four NPs varied significantly. The amounts of albumin, transferrin and α-1 antitrypsin were greater in the coronas of BaSO4 and ZnO than that of the two CeO2 NPs. The uptake of BaSO4 in AMs was less than CeO2 and silica-coated CeO2 NPs. No identifiable ZnO NPs were observed in AMs. Gold NPs coated with albumin or citrate instilled into the lungs of rats acquired the similar protein coronas and were cleared from the lungs to the same extent. Conclusions: We show that different NPs variably adsorb proteins from the lung lining fluid. The amount of albumin in the NP corona varies as does NP uptake by AMs. However, albumin coating does not affect the translocation of gold NPs across the air-blood barrier. A more extensive database of corona composition of a diverse NP library will develop a platform to help predict the effects and biokinetics of inhaled NPs.
Engineered nanoparticles; Biokinetics; Nanotoxicity; Protein corona; Lung macrophage
Human exposures to industrially-relevant nanoparticles
(NPs) are mainly by inhalation and are increasing in
occupational and environmental settings and from releases
from various nano-enabled products across their lifecycle
]. Airborne NPs deposited in respiratory tract airways
are cleared by the mucociliary apparatus; others reach the
alveolar regions of the lung where they interact with the
alveolar lining fluid. A very few may pass through the layer
of pneumocytes and translocate to blood where they may
reach other organs [
]. Most particles deposited in the
distal lung are ingested by lung macrophages and
ultimately dissolve [
In alveolar spaces, corona formation takes place in
the alveolar lining fluid. It consists of plasma proteins,
a surface-active phospholipid (PL)-protein mixture, also
known as pulmonary surfactant, and a thin layer of
aqueous hypophase especially in the alveolar “corners”
]. Pulmonary surfactant is composed of 85–90% (w/w)
PLs and 10% (w/w) of surfactant-specific proteins, such as
the hydrophilic surfactant proteins A (SP-A) and D
(SP-D). The hydrophobic surfactant proteins B (SP-B)
and C (SP-C) are present to a lesser extent (<5% of the
surfactant proteins, w/w) [
]. Studies have shown a
pivotal role for surfactant proteins in lung host defense.
SP-A and SP-D opsonize inhaled microbes, allergens,
and other foreign bodies such as NPs to varying
degrees, and promote their recognition, ingestion and
dissolution by resident alveolar macrophages (AMs)
and other leukocytes [
NP surface charge, covalent/coordinate bonding and
hydrogen bonding propensity are important NP surface
properties that may influence their adsorption of lipids
and proteins [
]. The effects of hydrophobicity and
surface charge of engineered nanoparticles (ENPs) on
their binding to hydrophobic, positively-charged proteins
SP-B and SP-C have been reported [
]. It was found
that anionic nanoparticles selectively adsorbed SP-B1–25,
but not SP-C. Recent studies showed that inhaled diesel
exhaust NPs exhibit a corona of SP-A, SP-D, and
]. The role of albumin in the translocation of
intravenously injected gold NPs (AuNPs) to different
organs has been described [
]. However, albumin’s
influence on translocation across the air-blood barrier is
unknown. Schleh et al. studied the effect of SP-D on the
translocation of Au NPs (20 nm) from the lungs to the
]. They reported that SP-D had only a
minor effect on early Au NP translocation.
Adsorption of biomolecules on to the NP surface occurs
rapidly and results in the formation of a
phospholipidprotein ‘corona’ on the NP surface [
]. The determinants
of corona composition are underappreciated and poorly
characterized, but are critical in determining the
subsequent fate and effects of NPs in both in vitro and in vivo
systems. It is reasonable to assume that the interactions of
NPs with lung cells occur with the NP-protein
phospholipid complex and not with a pristine NP surface. The
protein corona thus formed can significantly affect the
manner in which lung cells interact with, recognize, and
process NPs [
]. The influence of the NP corona on the
subsequent responses of the lungs to NPs needs to be
Translocation of gold, silver, TiO2, polystyrene, and
carbon-based particles across the air-blood barrier into
the circulation and extrapulmonary organs, although small
and poorly understood, has been described in a review
]. We have performed pulmonary toxicological and
biokinetic studies of cerium oxide (CeO2), barium sulfate
(BaSO4) and zinc oxide (ZnO) NPs [
]. We also
studied the consequences of a surface-modified CeO2 with a
nanothin amorphous SiO2 coating (Si-CeO2) on clearance
of the core cerium NPs [
]. In these studies, we explored
whether radiolabeled 141CeO2, Si-141CeO2, 131BaSO4 and
65ZnO NPs of similar size differ in lung clearance after
intratracheal (IT) instillation. We showed that the
pulmonary clearance was 65Zn > 131Ba > 141Ce
(SiCeO2 > 141Ce (CeO2) [
]. Of the four NPs examined,
65Zn cleared the lung fastest most likely due to high
dissolution of 65ZnO NPs [
]. Among the other three NPs,
131Ba cleared the lung fastest (84% of the instilled dose
cleared the lung by day 28). 141Ce from silica-coated
141CeO2 was cleared from the lungs relatively faster than
from uncoated 141CeO2 [
]. These differences may be
influenced by how these NPs interact with the lung lining
fluid resulting in corona formation which in turn
determine the NP fate and biological effects.
As the corona may also modulate the overall NP
biokinetics and biological effects, we now further
characterize the physicochemical properties of CeO2,
Si-CeO2, BaSO4 and ZnO NPs. These properties
include surface charge, agglomeration in various fluids,
and the composition of the protein corona formed
when incubated in concentrated bronchoalveolar lavage
fluid (BALf ). Finally, we tested the role of albumin, the
major protein in the lung lining fluid, on the clearance
of instilled gold NPs in rats and on the acquisition of
protein corona when incubated in BALf.
Nanoparticle physicochemical characterization
The synthesis of CeO2, Si-CeO2 and ZnO NPs used in
this study is outlined in detail in our previous
publications and summarized in the methods section. BaSO4
NPs (NM-220) were obtained from BASF SE
(Ludwigshafen, Germany). Transmission electron micrographs of
NPs suspended in deionized (DI) water are shown in
Fig. 1. Table 1 lists selected physicochemical
characteristics of the NPs. The primary NP sizes were similar (29
to 36 nm). The surface areas were identical for the two
CeO2 NPs, while the BaSO4 and ZnO NPs had higher
specific surface areas. The hydrodynamic diameter of NP
suspensions in DI and zeta potentials were measured
before incubation in harvested cell-free rat BALf for 30 min
at 37 °C by dynamic light scattering (DLS) using a
Malvern ZetaSizer Nano (Westborough, MA). The NPs
were incubated in BALf to examine the formation of the
protein corona and its influence on NP agglomerate size
and zeta potential. The multiple washing steps removed
unbound and loosely bound proteins from the NPs. The
zeta potentials of CeO2 and ZnO NPs changed from
positive to negative, and the hydrodynamic diameters of
all NPs increased after incubation in BALf. After the
acquisition of NP coronas, the conductance of the NP
suspensions in DI water increased dramatically (Table 1).
Protein corona characterization
We analyzed the composition of the acquired NP
protein corona using methods described previously
]. We performed 1-D SDS-PAGE and Coomassie
staining of total proteins detached from the NPs to
visualize the corona components on gel bands. The
proteins in the excised gel bands were identified in an
LTQ Orbitrap Velos Pro ion-trap mass spectrometer.
The stained SDS-PAGE gel and the relative amounts of
identified proteins after NPs were incubated with BALf
are shown in Fig. 2. We found that the protein
composition of the NP coronas were different. The total
amount of protein bound per unit mass (μg/mg NPs)
was in the order of CeO2 < Si-CeO2 < BaSO4 < ZnO
(Fig. 2b). The amounts of individual proteins in the
corona are shown in Table 2. When expressed as % of
total bound protein, albumin, transferrin and
α-1antitrypsin were relatively higher in the corona of BaSO4
and ZnO than that of CeO2 and Si-CeO2 NPs (Fig. 2c).
When expressed as bound proteins per surface area
(μg/m2), ZnO NPs acquired greater amounts of
transferrin, albumin and α-1-antitrypsin and BaSO4 NPs
acquired greater amounts of albumin than did both
CeO2 and Si-CeO2 NPs (Fig. 2d).
Nanoparticle uptake by alveolar macrophages
To explore if AM uptake of CeO2, Si-CeO2, BaSO4 and
ZnO NPs in vivo were different, we instilled rats
intratracheally with NP suspensions at a dose of 1 mg/kg
body weight. Then, we measured the degree of
phagocytosis of these NPs by AMs recovered from BALf at
24 h. Using transmission electron microscopy (TEM),
we found that the % of sections of AMs with internalized
BaSO4 NPs was less than CeO2 and Si-CeO2 (Fig. 3).
No identifiable ZnO NPs were observed. An additional
TEM examination at 1-h post-instillation also did not
show ZnO NPs in lavaged cells (data not shown).
Role of albumin in translocation and AM uptake of gold nanoparticles
Our results showed that levels of some proteins in the
NP corona, such as transferrin, albumin and A1AT
(α1antitrypsin), varied among the 4 NPs. As shown in Fig.
2c, BaSO4 and ZnO NPs had the highest level of
albumin in their corona. Therefore, we tested the influence
of albumin coating on the fate of instilled gold (Au) NPs
since it was the major protein observed in NP coronas.
We coated Au NPs (core size 18 ± 2 nm) with either
albumin or citrate and studied their translocation across
the air-blood barrier. Fig. 4a shows a TEM image of
citrate-coated Au NPs and Table 3 shows hydrodynamic
diameter and zeta potential of citrate- or human serum
albumin-coated Au NPs in deionized water and in BALf.
The hydrodynamic diameter in deionized water was
25 ± 0.2 nm (citrate-coated Au NPs) and 53 ± 0.1 nm
(albumin-coated Au NPs). When incubated in BALf, the
hydrodynamic diameter of citrate- and albumin-coated
Au NPs changed from 25 to 275 nm and 53 to 115 nm,
respectively. There was also a change in negative zeta
potential value of the albumin-coated Au NPs (−14 to
−56 mV). Fig. 4b shows the absorption spectra of
citratecoated Au NPs in water (black curve) and of
albumincoated Au NPs in PBS.
First, we examined the composition of acquired
protein corona formed on the two Au NPs after
incubation in BALf. We found no significant qualitative or
quantitative differences in the corona composition
between citrate- and albumin-coated Au NPs (Fig. 5).
We found that in both cases, the corona primarily
We then IT-instilled rats with either citrate- or
albumin-coated Au NPs and examined the Au content
in lungs and other selected organs at 6 and 24 h
postinstillation. Our results showed that there was no
significant difference in the fate of the two Au NPs
(Fig. 6). We examined the Au content in the liver,
spleen, the major organs of the mononuclear
phagocyte system and tracheobronchial lymph nodes.
Extrapulmonary retention of Au from both NPs was in the
order liver > lymph node > spleen. The translocation
of Au to the spleen was lower in rats instilled with
albumin-coated Au NPs (0.001%) than in rats instilled
with citrate-coated Au NPs (0.003%). The difference
was statistically significant at 24 h post-instillation
Our data showed that the resulting coronas of both
Au NPs in vitro were predominantly composed of
albumin, transferrin and Tubb2A, and were not different
between the two Au NPs (Fig. 5). However, the increase
in hydrodynamic size of albumin-coated Au NPs was
two-fold compared to ten-fold of the citrate-coated Au
NPs (Table 3). The zeta potential significantly changed
only with albumin-coated Au NPs (−14 to −56 mV).
This can be attributed to additional proteins from BALf.
Also, the conductance of the suspending medium
increased moderately with albumin-coated compared to
citrate-coated Au NPs (2.2 vs. 3.6 fold increase). To test
if AM uptake of citrate- and albumin-coated Au NPs
were different, we also measured the uptake of these
NPs in AMs recovered from rats 24 h after instillation.
We found that the percentage of macrophage sections
with internalized Au NPs was not different between rats
instilled with citrate- and albumin-coated Au NPs
(Fig. 7). However, macrophages that have taken up
albumin-coated Au NPs had fewer endosomes with
densely packed NPs compared to macrophages with
citrate-coated Au NPs.
Our goal was to characterize the physicochemical
properties of CeO2, Si-CeO2 BaSO4 and ZnO NPs when
suspended in distilled water and after incubation in
harvested cell-free rat BALf for 30 min at 37 °C. We
focused on the protein components of the NP corona; the
analysis of the phospholipid component will be reported
elsewhere. CeO2, Si-CeO2, BaSO4 and ZnO NPs were
incubated in BALf to determine the formation of the
protein corona, and its relation to their agglomerate size
and zeta potential. After incubation in BALf, CeO2 and
ZnO NP zeta potentials changed from positive to
negative, and the hydrodynamic diameters and conductance
of all four NP suspensions increased. Changes in zeta
potential, conductance and agglomerate sizes were likely
due to acquisition of protein and phospholipid corona
from the BALf [
Our analyses showed differences in the amounts of
adsorbed proteins per mg of NPs (Fig. 2b) and surface
area (Fig. 2d). Corona proteins were compared based
on NP mass and surface area since normalization
based on particle number is complicated because of
NP agglomeration in liquid media. Moreover, it is
difficult to determine whether these proteins adsorb
primarily on agglomerate surfaces or can also penetrate
the ‘agglomerates’ and coat individual particles.
Differences in the corona proteins among the four
NPs may be due to the differences in NP surface
chemistry, charge and hydrophobicity that in turn influence
NP-biomolecular interactions. The surface chemistry of
nanoparticles influences their agglomeration and the
formation of coronas around them. It is thought that
NPs may aggregate first and then form a corona around
the aggregate, or may form coronas around individual
particles first and then aggregate. Both aggregation and
corona formation are natural processes that lower the
surface energy of NPs [
Among the corona proteins, amounts of albumin,
transferrin and α-1-antitrypsin were relatively greater in the
coronas of BaSO4 and ZnO than of CeO2 and Si-CeO2 NPs
(Fig. 2c, d). These proteins are among those that can cross
the alveolar-epithelial barrier [
transport in the alveolar epithelium have been reported
for albumin and transferrin [
]. Translocation of organic
NPs conjugated with human serum albumin has been
]. However, little is known about the
precise role of these biomolecules in pulmonary clearance
1.21 ± 0.17#
1.20 ± 0.26#
Alveolar macrophages are key resident phagocytic cells
of the lungs that sequester and bioprocess NPs as well
as orchestrate inflammatory responses [
internalized in AMs, NPs are slowly cleared from the
lungs by particle dissolution and cell migration. The rate
at which AMs phagocytize NPs can be influenced
significantly by the composition of the corona . Coating
magnetite and TiO2 with SP-A increases their uptake in
macrophages suggesting that the NP protein corona can
affect particle recognition, phagocytosis, and processing
by AMs [
We studied AM uptake of CeO2, Si-CeO2, BaSO4 and
ZnO NPs in vivo using transmission electron microscopy
(TEM) on lavaged lung cells at 24 h post-instillation. As
shown in Fig. 3, the % of AM sections with internalized
BaSO4 NPs was less than that of CeO2 and Si-CeO2 NPs.
Variations in macrophage uptake of NPs may have been
influenced by their surface characteristics and the
resulting corona. There were no identifiable ZnO NPs. Even at
0.46 ± 0.19
0.48 ± 0.25#
0.60 ± 0.39#
0.74 ± 0.45*
0.72 ± 0.49*
0.65 ± 0.40*
0.80 ± 0.46*
0.72 ± 0.38*
4.17 ± 1.73
0.16 ± 0.07
0.17 ± 0.09
0.22 ± 0.14*
0.26 ± 0.16*
0.26 ± 0.18@
0.29 ± 0.16
0.26 ± 0.14
1.49 ± 0.62
1-h post-instillation, ZnO NPs were not observed in
lavaged cells (data not shown). The absence of
recognizable ZnO NPs in lavaged cells suggests both
rapid ingestion by macrophages and fast dissolution
due to greater solubility [
]. It is also possible that the
release of Zn ions inhibits further ZnO NP uptake by
macrophages in vivo.
Our data on proteins in particle coronas showed that
transferrin, albumin and A1AT varied significantly
among the four NPs. We tested the influence of
albumin coating on the fate of instilled Au NPs since it was
the major protein observed in all NP coronas. Because
Au particles are highly insoluble, we chose them to
study the role of albumin in NP translocation from the
lungs. In addition, Au NPs can be surface
functionalized with organic ligands. Although initially coated with
citrate or albumin, Au NPs would likely acquire
coronas as they interacted with the lung lining fluid in
vivo. Therefore, we examined their protein coronas
after incubation in BALf. As shown in Fig. 5, the
coronas of both Au NPs predominantly consisted of
albumin. The extent to which the apparent higher amount
of albumin in the albumin-coated NPs included the
original albumin coating could not be determined. Our
pharmacokinetic data showed no significant difference
in clearance of the two Au NPs from the lungs. Only
0.1 to 0.4% Au NPs translocated from the lungs to
extrapulmonary organs. This was consistent with minimal
translocation observed in previous studies [
19, 33, 34
all organs examined, Au retention in the spleen at 24 h
was the only significant difference. It was lower in rats
instilled with albumin-coated Au NPs (0.001%) than with
citrate-coated Au NPs (0.003%). This small but statistically
significant difference in spleen retention needs further
Our data showed that even with initial coating of
citrate or albumin of Au NPs, the resulting coronas
formed when incubated in vitro did not differ. They
were mainly composed of albumin, transferrin and
Tubb2A. The increases in conductance of Au NP
suspensions after corona acquisition were similar.
However, the increase in hydrodynamic size of
albumincoated Au NPs after incubation in BALf was two-fold
compared to ten-fold of the citrate-coated Au NPs
(Table 3). The zeta potential changed significantly only
with albumin-coated Au NPs (−14 mV to −56 mV).
This change in zeta potential might be related to the
smaller increase in agglomerate size of albumin-coated
Au NPs after acquisition of corona. Hydrodynamic
diameters were greater likely due to NP agglomeration,
acquisition of protein and phospholipid corona or both.
The changes in hydrodynamic diameters of NPs may be
explained by competing reactions of NPs with ions that
promote agglomeration and with proteins and lipids
Fig. 4 a Transmission electron micrograph of citrate-coated Au NPs. b UV-vis spectra of citrate-coated Au NPs in water (black curve) and
albumin-coated Au NPs in PBS (red curve). The Au NPs show a red-shift in the peak absorbance wavelength after albumin coating
that of citrate-coated Au NPs. The difference might be
due to greater dissolution of BaSO4 than Au NPs in
phagolysosomes during the 24-h post-instillation period.
As we found that both the protein corona and the lung
clearance of the two Au NPs were not different, it let us
consider the corona formation on functionalized or
surface-modified NPs that occurs in in vivo experiments.
Additional studies are needed to explore the potential
role of other corona proteins as well as phospholipids in
the fate of NPs in the lungs and in extrapulmonary sites.
We hypothesize that the interaction of NPs with
biomolecules of the lung lining fluid at the air-blood barrier
leads to the formation of an NP corona, which may
affect their uptake by AMs and other cells as well as
their translocation across the air-blood barrier.
Our data show that different NPs bind significantly
different proteins as they interact with lung lining fluids.
Furthermore, we showed that gold NPs coated with
citrate or albumin acquired similar protein coronas, were
cleared from the lungs, and were retained in
extrapulmonary organs to the same degree. The extent to which
the protein corona is a critical determinant of the fate
and biological effects of NPs remains to be established.
Studies examining the independent and collective
effects of the major proteins and phospholipids of lung
lining fluid forming the NP corona, and their role in
regulating translocation of NPs across the air blood
barrier and in determining which cells bind and/or
ingest NPs, are warranted. We believe that an extensive
database of both protein and phospholipid coronas of a
forming the corona that prevent agglomeration. The
total amounts of recovered corona proteins from Au
NPs were not significantly different.
We explored if the observed changes in agglomerate
sizes and zeta potentials when Au NPs interact with
lung lining fluid in vivo also affect their uptake by
AMs. The fraction of macrophage sections with
internalized Au NPs was about 2.5 times higher than with
CeO2 NPs (Figs. 3 and 7) suggesting that the corona
with more albumin might promote phagocytosis, as
shown previously [
]. However, fewer macrophage
sections contained internalized BaSO4 NPs although their
corona had equivalent amounts of albumin compared to
diverse NP library will help predict the pulmonary
effects and biokinetics of inhaled NPs.
Nanoparticle synthesis and characterization
Synthesis of nanoparticles
Uncoated CeO2, Si-CeO2 and ZnO NPs were made by
flame spray pyrolysis using the Versatile Engineered
Nanomaterial Generation System (VENGES) at Harvard
]. Detailed physicochemical and
morphological characterization of these NPs was reported earlier
]. BaSO4 NPs (NM-220) were obtained from
BASF SE (Ludwigshafen, Germany). It was a reference
material for the Nanomaterial Testing Sponsorship
Program of the Organization for Economic Cooperation
and Development (OECD). The characterization of the
original batch distributed as NM-220 was published
Monodisperse Au NPs were prepared with
modifications of a previously published protocol [
]. Au NPs
were produced by mixing 5.3 mg of NaAuCl4 * 2H2O
dissolved in 25 mL of Milli-Q grade water with 1 mL of
a 1% trisodium citrate solution. Then glass beads were
added for mixing and heat distribution. The mixture
was boiled in a microwave for 90 s. The deep-red
suspension was cooled down slowly to room temperature
and stored protected from light at room temperature.
An aliquot of prepared Au NPs were functionalized
with human serum albumin (HSA) with few
modifications from a previously described method [
Fig. 7 Transmission electron micrographs of lavaged cells at 24 h
from rats instilled with Au NP suspensions at a dose of 1 mg/kg
body weight. a Macrophage uptake of citrate-coated Au NPs and
b albumin-coated Au NPs. Micrographs in 6A and 6B insets are
higher magnification of the areas shown. c Morphometric analysis
of NP uptake. Macrophage uptake was scored similarly as described
in Fig. 3. No significant difference in macrophage uptake of NPs
(n = 185, citrate-coated Au NPs, n = 191, albumin-coated Au NPs)
was found. Data are mean ± SE of % of macrophages (n = 3 rats
1 mL of colloidal Au NPs at 5 nM was added drop-wise
under constant stirring to 500 mL of HSA solutions at
5–10 μM. After incubation for 40 min at room
temperature in the dark, the solution was centrifuged
for 6 min at 14,000 rpm. The supernatant was removed,
and the NPs were washed twice by
centrifugation/resuspension in Milli-Q water and finally dispersed in a
PBS solution. The size, shape and monodispersity of
the NPs were verified by transmission electron
microscopy. UV-Vis measurements were performed with
an Ultro spec 2100 Pro (Amersham). The maximum
absorption of Au NPs is centered at 520 nm (without
HSA) and at 525 nm (with HSA). The hydrodynamic
diameter (DH), polydispersity index (PdI), and zeta
potential (ζ) of each Au-NP suspended in DI water were
measured by DLS using a Zetasizer Nano-ZS (Malvern
Instruments, Worcestershire, UK). The DLS
measurements were performed in ion-free conditions since the
citrate-coated Au NPs aggregated in PBS. However, the
stability of the AuNPs in ionic solutions increased once
the NPs were coated with either albumin alone (data
not shown) or after acquisition of protein corona.
Hydrodynamic diameter and zeta potential
CeO2, silica-coated CeO2, BaSO4 and ZnO NPs
suspensions at specified concentrations in sterile distilled
water were sonicated in conical polyethylene tubes. A
critical dispersion sonication energy (DSEcr) to achieve
the smallest particle agglomerate size was used, as
previously reported [
]. The suspensions were sonicated
at 242 J/ml (20 min/ml at 0.2 watt power output) in a
cup sonicator fitted on a Sonifier S-450A (Branson
Ultrasonics, Danbury, CT). The sample tubes were
immersed in running cold water to minimize heating of
the particles during sonication. The hydrodynamic
diameter (DH), polydispersity index (PdI), and zeta potential (ζ)
of each suspension were measured by DLS using a
Zetasizer Nano-ZS (Malvern Instruments, Worcestershire,
UK). The Au NP suspensions were similarly analyzed
without prior sonication.
Characterization of NPs after incubation in BALf: Protein
corona and dynamic light scattering analysis
A total of 9 ml pooled BAL fluid from 3 rats (3 ml/rat)
was centrifuged at 350 x g to remove cells. Then, CeO2,
Si-CeO2, BaSO4 and ZnO (200 μL of 1 mg/mL) were
incubated in 3 mL BALf for 30 min at 37 °C. We chose
30min incubation since corona formation in the lungs
occurs soon after the particles interact with the alveolar
lining, and because those early events are relevant to their
fate in the lungs [
After incubation, the NP suspensions were centrifuged
for 10 min at 14,500 x g. The resulting pellet was washed
in DI water three times. The use of DI water avoided the
potential interaction of hydrophobic phospholipid
components of the BALf with the corona proteins. The pellets
containing NPs with ‘hard corona’ were suspended in
20 μL of DI water. One set was used for DLS analyses and
the other set was used for corona protein composition
The NP pellets for corona protein analyses were also
suspended in 20 μL of DI water to which 10 μL of 4×
Laemmli sample buffer was added and vortexed. The
sample was then heated to 95 °C for 7 min. After
cooling to room temperature, 6 μL of mixed solution
(57 μL Laemmli and 3 μL β-mercaptoethanol was
added to 18 μL of the sample. The samples were then
loaded onto a gel and proteins were visualized by 1D
SDS-PAGE in combination with Coomassie staining.
The protein corona experiment was repeated three
times (total of 3 rats/NP) for CeO2, Si-CeO2, BaSO4
and ZnO, and two times for citrate-coated Au and
albumin-coated Au NPs (total of 4 rats/NP).
Distinct bands from 2 gels were excised and subjected
to a modified in-gel trypsin digestion procedure [
Peptides were later extracted and then dried in a
SpeedVac (~1 h). The samples were then stored at 4 °C until
analysis. Samples were analyzed at the Harvard Medical
School Taplin mass spectrometry facility (Boston, MA).
On the day of analysis, the samples were reconstituted
in 5–10 μL of HPLC solvent A (2.5% acetonitrile, 0.1%
formic acid). A gradient was formed and peptides were
eluted with increasing concentrations of solvent B
(97.5% acetonitrile, 0.1% formic acid) [
peptides were subjected to electrospray ionization and then
analyzed in an LTQ Orbitrap Velos Pro ion-trap mass
spectrometer (Thermo Fisher Scientific, San Jose, CA).
Peptides were detected, isolated, and fragmented to
produce a tandem mass spectrum of specific fragment ions
for each peptide. Peptide sequences (and protein
identity) were determined by matching protein databases
with the acquired fragmentation pattern by the software
program, Sequest (ThermoFisher, San Jose, CA) [
Spectral matches were manually examined and multiple
identified peptides per protein were required. The
relative amounts of identified proteins were calculated
based gel band densities obtained with ChemiDoc™
XRS System (BioRad, Hercules, CA) and analyzed with
The protocols in this study were approved by the Harvard
Medical Area Animal Care and Use Committee (Boston,
MA). Fifty two male Wistar Han rats (8 weeks old) were
obtained from Charles River Laboratories (Wilmington,
MA) and were housed in standard microisolator cages
under controlled conditions of temperature, humidity, and
light at the Harvard Center for Comparative Medicine.
The rats were fed commercial chow (PicoLab Rodent Diet
5053, Framingham, MA) and provided with
reverseosmosis purified water ad libitum. The animals were
acclimatized in the facility for 7 days before the start of each
experiment. These animals were used for the experiments
Assessment of uptake of NPs in vivo by alveolar macrophages
CeO2, Si-CeO2, BaSO4, ZnO, citrate-coated Au and
albumin-coated Au NP suspensions were instilled in
separate cohorts of rats (n = 3 per NP) at a dose and
concentration of 1 mg/kg and 0.67 mg/ml. At 24 h
postinstillation, rats were sacrificed and their lungs were
lavaged, as described previously [
]. BAL cells were
cytocentrifuged and fixed in 2.5% glutaraldehyde in HEPES
buffer, pH 7.4. The pellets were processed for electron
microscopy. Uptake of NPs by cells was analyzed in a JEOL
1400 transmission electron microscope (JEOL USA, Inc.,
Peabody, MA). Random micrographs from each rat were
scored for the presence of internalized NPs in each
macrophage. Macrophage uptake was scored as +, ++, or
+++ when 1–2, 3–4 or ≥5 particle-containing phagosomes
were observed in randomly selected electron micrographs,
respectively. Alveolar macrophages were scored (CeO2
n = 248, Si-CeO2 n = 273, BaSO4 n = 295, ZnO n = 250,
citrate-coated Au n = 185, albumin-coated Au n = 191).
Pharmacokinetics of Au after intratracheal instillation of Au NPs
Fifty microliters of each NP suspension were added to
sterile distilled water to prepare a volume dose of 1.5 ml/
kg body weight. Before dosing, each rat was anesthetized
with isoflurane (Piramal Healthcare, Bethlehem, PA).
Based on ICP-MS analysis, the final dose was 37 μg Au/
rat (citrate-coated Au NPs) and 90 μg Au/rat
(albumincoated Au NPs). The NP suspension was intratracheally
instilled into the lungs of 12 rats per Au NP. Then, six rats
from each group were euthanized at 6 h and 24 h
postdosing. They were anesthetized with vaporized isoflurane,
and exsanguinated via the abdominal aorta. The lungs,
tracheobronchial lymph nodes, spleen, and liver were
collected for Au analysis using ICP-MS. Data were
expressed as percentage of the administered dose retained
in each organ.
All data were analyzed using multivariate analysis of
variance (MANOVA) followed by Tukey post hoc tests
using SAS Statistical Analysis Software (SAS Institute,
Cary, NC). The macrophage uptake of Au NPs in vivo
was analyzed using Student's t test.
A1AT: α1-antitrypsin; AM: alveolar macrophage; Au NPs: gold nanoparticles;
BALf: bronchoalveolar lavage fluid; BET: Brunauer–Emmett–Teller;
C3: complement component 3; Cfb: complement factor B; DH: hydrodynamic
diameter; DI: deionized water; DPBS: Dulbecco’s phosphate buffered saline;
DSEcr: critical dispersion energy; Dxrd: primary particle size based on X-ray
diffraction; ICP-MS: inductively coupled-mass spectrometry; LC-MS: liquid
chromatography-mass spectrometry; MW: molecular weight;
NP: nanoparticle; PdI: polydispersity index; PL: phospholipid; PLUNC: palate,
lung, nasal epithelium clone protein; SDS-PAGE: sodium dodecyl sulfate
polyacrylamide gel electrophoresis; SP-A: surfactant protein A;
SPB: surfactant protein B; SP-C: surfactant protein C; SP-D: surfactant protein D;
SSA: specific surface area; TEM: transmission electron microscopy;
Tf: transferrin; TiO2: titanium dioxide; Tubb2A: tubulin Beta 2A Class IIa;
UVvis: ultraviolet-visible spectrophotometry; UV-Vis: visible and ultraviolet
spectroscopy; ζ: zeta potential; ρ: density
We kindly acknowledge the financial support from the National Institutes
of Environmental Health Sciences (NIH-P30ES000002, K99ES025813 and
1U24ES026946). The authors also gratefully acknowledge the technical help
of Dr. Ross Tomaino and Dr. Anoop Pal, and Melissa Curran for editorial advice.
The study was funded by the National Institutes of Environmental Health
Sciences (NIH-P30ES000002 and 1U24ES026946) and the National Science
Foundation (1530767). NVK is supported by NIEHS grant K99ES025813.
Availability of data and materials
The datasets supporting the conclusion of this article are included within the
article. There are 7 figures and 3 tables. All relevant raw data are freely
available to researchers wishing to use them.
NVK, RMM, AS, WK, SK and JDB designed, performed and evaluated the
experimental results. AS, PL and FD performed protein corona evaluation. PD
and GP synthesized and characterized CeO2, Si-CeO2 and ZnO NPs. PA and SK
synthesized and characterized the Au NPs. TCD performed statistical analyses.
NVK, RMM, WK, SK and JDB wrote this manuscript. All authors read and
approved the final manuscript.
All animal experiments are in compliance with protocols approved by the
Harvard Medical Area Animal Care and Use Committee (Boston, MA).
Consent for publication
All authors read, corrected, and approved the manuscript.
The authors declare no competing financial interest.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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