Interaction of Fine Particles and
Nanoparticles with Red Blood Cells
Visualized with Advanced
B A R B A R A M . R O T H E N - R U T I S H A U S E R , *, ‡
S A M U E L S C H U ¨ R C H ,§B E A T H A E N N I ,‡
N A D I N E K A P P ,|A N D P E T E R G E H R‡
Institute of Anatomy, Division of Histology, University of Bern,
Bern, Switzerland, Department of Physiology and Biophysics,
University of Calgary, Calgary, Alberta, Canada, and
Department of Veterinary Anatomy, University of Bern,
So far, little is known about the interaction of nanoparticles
with lung cells, the entering of nanoparticles, and their
transport through the blood stream to other organs. The
of differing materials and of different charges were
studied in human red blood cells. As these cells do not
We combined different microscopic techniques to visualize
fine and nanoparticles in red blood cells: (I) fluorescent
particles were analyzed by laser scanning microscopy
combined with digital image restoration, (II) gold particles
were analyzed by conventional transmission electron
microscopy and energy filtering transmission electron
by energy filtering transmission electron microscopy. By
using these differing microscopic techniques we were able
in red blood cells. We found that the surface charge
and the material of the particles did not influence their
entering. These results suggest that particles may penetrate
the red blood cell membrane by a still unknown mechanism
different from phagocytosis and endocytosis.
There is evidence from a number of epidemiological studies
that particulate matter causes adverse health effects associ-
(1, 2). Recent studies indicate a specific toxicological role of
nanoparticles (3). Thus particles a few nanometers in
In addition to the generation from combustion processes of
from other sources, especially those related to nanotech-
nological processes (4, 5). Nanoparticles have unique chemi-
cal, physical, and electrical properties. They behave unlike
particles and solutes. New properties emerge that are not
exhibited by larger particles having the same chemical
composition. These properties include different colors and
different electronic, magnetic, and mechanical properties,
any or all of which may be altered at the nanoscale. It is very
important to collect risk data, in particular health risk data,
so that questions can be answered and problems can be
addressed during the early stage of the development of the
new technologies (6). Most of the concerns regarding
inflammatory and toxic than fine particles (7).
Not only is the effect of nanoparticles on human health
an important issue, but also in particular the mechanisms
involved in the penetration of the human body by these
particles. The wall of the respiratory system consists of a
series of barrier components that protect against foreign
material. These include the surfactant film (8-10), the
mucociliary system (11), highly phagocytic airway macro-
phages (12-14), and the epithelium with its tight junctions
(15). However, despite the existence of these barriers,
respiratory diseases are frequent and increasing (16) and
PM10) deposited on the airways are displaced into the
subphase below the surfactant film and may be coated with
process (17, 18). As a result of the displacement, particles
come into contact with the epithelium and airway macro-
phages (9, 17, 19). Not all antigens are phagocytosed by the
macrophages; many of them are able to cross the air-blood
barrier of the lung and may enter the circulatory system (20)
as it also has been shown for nanoparticles (21) and for
respiratory pathogens (22).
or adhesive interactions (21). In the present study the
interaction of different nanoparticles with cells was inves-
tigated to examine the influence of different materials and
of different charges for the entering. Human red blood cells
(RBC) were used as a model for nonphagocytic cells as they
do not have phagocytic receptors, which are found on the
surface of professional phagocytes (for a review see 23). The
integral membrane proteins and the underlying membrane
skeleton (for a review see 24).
Since nanoparticles have a size of less than 0.1 µm their
identification is difficult. We have applied different micro-
(1) laser scanning microscopy (LSM) in combination with
digital data restoration to visualize fluorescently labeled
microscopy (TEM) to identify electron dense nanoparticles
such as 0.025 µm gold particles, and (3) energy filtering
nanoparticles, like, e.g., 0.02-0.03 µm TiO2 particles. We
found that neither the surface charge nor the material of the
These results suggest that the entering mechanism of
* Corresponding author e-mail: email@example.com;
phone: ++41-31-631-8441; fax: ++41-31-631-3807.
†This paper is part of a focus group on Effects of Nanoparticles.
‡Institute of Anatomy, Division of Histology, University of Bern.
§University of Calgary.
|Department of Veterinary Anatomy, University of Bern.
Environ. Sci. Technol. 2006, 40, 4353-4359
10.1021/es0522635 CCC: $33.50
Published on Web 04/05/2006
2006 American Chemical Society VOL. 40, NO. 14, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY94353
Materials and Methods
(containing 25 mM Hepes, LabForce AG, Nunningen, Swit-
zerland) with 10% heat inactivated (pooled) human serum
(Blood Donation Service), 1% L-glutamine (LabForce), and
Switzerland) in two-chamber slides (VWR International AG,
were incubated with particles for 4-24 h.
Particle Incubation. Commercially available particles
were used: Fluoresbrite plain yellow-green microspheres
with diameters of 1 and 0.2 µm (Polysciences, Chemie
Brunschwig AG, Basel, Switzerland); fluorescent particles,
Brunschwig AG, Basel, Switzerland); FluoSpheres, carboxy-
modified microspheres, yellow-green with diameters of 1,
0.2, and 0.02 µm (Molecular Probes, Invitrogen AG); Fluo-
Spheres, amino-modified microspheres, yellow-green with
diameters of 1 and 0.2 µm (Molecular Probes, Invitrogen
AG); Cationic and BSA gold tracers with a diameter of 0.025
µm (Aurion, Anawa Trading SA, Wangen, Switzerland); and
titanium (IV) oxide (TiO2), anatase 99.9% (metal basis) with
a mean diameter of 0.02-0.03 µm (Alfa Aesar, Johnson
Matthey GmbH, Karlsruhe, Germany).
All particle dilutions were sonicated for 2 min prior to
incubation with cells in order to avoid aggregation. Fluo-
Gold particles were diluted in RPMI 1640 to a final dilution
of 6.6 × 1010particles mL-1. A stock solution of TiO2 in
Millipore water (2.5 mg mL-1) was diluted in RPMI to finally
was then added to cell cultures. Incubations were done for
LSM analysis RBC were fixed in 2.5% glutaraldehyde/PBS,
resulting in a red autofluorescence. Preparations were
mounted in PBS/glycerol (2:1) containing 170 mg mL-1
Mowiol 4-88 (Calbiochem, VWR International AG).
A MicroRadiance system from BioRad combined with an
inverted Nikon microscope (Eclipse TE3000, Lasers: HeNe
543 nm, and Ar 488 nm) was used. Image processing and
visualization was done using IMARIS, a 3D multichannel
image processing software for confocal microscopic images
visualization of particles at high resolution a deconvolution
algorithm was applied using the Huygens 2 software (Sci-
entific Volume Imaging B. V., Hilversum, Netherlands) in
noise. Deconvolution was done using the maximum likeli-
hood estimation algorithm method to effectively remove
noise and to recover details.
Transmission Electron Microscopy. For TEM analysis
RBC were centrifuged and the pellet was fixed with 2.5%
glutaraldehyde in 0.03 M potassium phosphate buffer, pH
M sodium cacodylate buffer, and with 0.5% uranyl acetate
in 0.05 M maleate buffer. Cells were then dehydrated in a
graded series of ethanol and embedded in Epon. Ultrathin
sections were cut and transferred on 200-mesh uncoated
copper grids, stained with uranyl acetate, counter-stained
with lead citrate according to standard methods (25), and
observed with a Philips 300 TEM at 60 kV (FEI Company
Philips Electron Optics, Zuerich, Switzerland).
Energy Filtering Transmission Electron Microscopy.
Ultrathin (e50 nm) sections were cut, mounted onto
uncoated 600-mesh copper grids, and stained with uranyl
acetate and lead citrate. The presence and localization of
TiO2 particles was investigated in a LEO 912 transmission
with an in-column energy filter allowing energy dispersion
for element specific contrast. TiO2particles were identified
by image-EELS (26). For elemental microanalysis, the L2,3
field and element-specific contrast for TiO2were obtained
by digital acquisition. Since gold was not suitable for
elemental analysis the gold particles were coated with silver
using silver enhancement reagent (AURION R-GENT SE-
the M4,5edge of Silver at 367 eV was used (27).
LSM Analysis. The entering of fine (1-0.2 µm) and nano-
particle (<0.1 µm) fluorescent microspheres was studied in
RBC as a model for nonphagocytic cells. For the localization
and visualization of the particles at high resolution a
deconvolution algorithm was applied to increase axial as
(amino-modified), and negatively charged (carboxy-modi-
fied) particles were used to investigate if the negatively
charged surface of RBC influence the entering of charged
and noncharged particles. Noncharged (Figure 1a), carboxy-
lated (Figure 1b), and positively charged (Figure 1c) 1 µm
particles were found to be attached to the cell surface but
were never seen inside the cells. This can also be demon-
strated with a shadow projection of the confocal data set
(Figure 1b inset). The amino-modified particles have a very
the green channel are visible in the red channel (Figure 1c),
however, it is very clear that the particles are not inside the
cells. In contrast, 0.2 µm negatively charged (Figure 1c) and
with the shadow projection image (Figure 1e inset). When
positively charged particles were used we found many
particles attached to the cell surface (Figure 1f), but we did
not see more particles inside the cells compared with non-
with digital data restoration even nanoparticles could be
visualized. Noncharged 0.078 µm particles (Figure 1e) and
negatively charged 0.02 µm particles (Figure 1f) were found
in RBC. Nanoparticles inside and attached to cells were
counted in a total of 25 cells. We found for the noncharged
particles 0.5 (SD 0.5) particles/cell within and 1.1 (SD 1.9)
particles 0.8 (SD 1.0) particles/cell inside, and 1.5 (SD 1.5)
modified nanoparticles available from Molecular Probes or
from Polyscience we used positively charged gold particles
(see next paragraph).
TEM Analysis. To obtain more information about the
entering and localization of nanoparticles in RBC, particles
with polystyrene fine and nanoparticles (0.2 and 0.078 µm
in diameter, respectively). Particles of 0.2 µm were found in
RBC (Figure 2a, a′). It was not possible to detect polystyrene
particles with a diameter of less than 0.1 µm in RBC because
Therefore, colloidal gold particles with a diameter of 0.025
µm were applied, a routinely used marker in electron
to RBC cultures. BSA gold particles were taken up by RBC
and found in the cytoplasma (Figure 2b, b′).
43549ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 40, NO. 14, 2006
to cultures to investigate if the positive charge enhances the
entering of particles. Entering of cationic gold particles has
been observed (Figure 2c, c′), however, we did not see more
particles inside RBC compared with noncharged and nega-
as for BSA gold particles. Larger agglomerates (> 0.2 µm)
were found to be attached to the cell membrane (Figure 2d).
EFTEM Analysis. Conventional TEM is not adequate for
the ultrastructural analysis of nanoparticles, since their size
and electron density are similar to artifacts due to specimen
preparation or the use of heavy metals for contrast enhance-
ment. Analytical TEM, which allows the resolution of the
chemical composition of structures, serves as an important
tool to investigate the interaction of nanoparticles with cells
at the ultrastructural level.
Therefore, RBC were incubated with ultrafine TiO2with
a mean diameter of 0.032 µm and investigated with EELS for
microanalysis. Ultrafine TiO2particles and small aggregates
it can also be seen that objects that look similar to TiO2
larger than 0.2 µm were seen attached to the membrane
(Figure 3b), but never within cells.
for specific elemental analysis of gold. However, the specific
M-edge of gold is at 2206 eV energy-loss. In this energy-loss
FIGURE 1. LSM micrographs of fluorescent polystyrene spheres attached to or within RBC. Autofluorescence of the cells is shown in
red, particles are green. The xy- and xz-projections allow the clear differentiation between intracellular (arrows) and extracellular
(arrowheads) particles. The images represent single optical sections (xy- and xz-projections). Yellow arrowheads mark the positions of
the projections. The inserts represent 3D reconstructions from the same data sets.
VOL. 40, NO. 14, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY94355
range, it is difficult to improve the signal-to-noise ratio due
to very low intensities. Since the elemental analysis of silver
(Figure 3c). Silver was also detected in larger complexes
attached to the cells or as free aggregates (Figure 3d).
On one hand the resolution of light microscopy is about 0.2
µm at best depending on the wavelength of the light. On the
other hand, by using TEM, although the resolution is much
higher, the particles are very difficult to identify, as the size
of the objects is similar to that of, for example, ribosomes.
By using a variety of advanced microscopic techniques we
were able to visualize nanoparticles with different surface
charges and of different material with light as well as with
transmission electron microscopy. By combining these
is significantly better than by using either method alone.
Using light microscopy we can study many cells in a short
or nanoenvironment) has to be examined with electron
can be studied.
Fine particles (1 and 0.2 µm) and nanoparticles (0.078
and 0.02 µm) which were fluorescently labeled and made of
a polystyrol material have been studied in RBC by LSM
combined with digital image restoration. To overcome
limitations caused by out-of-focus components resulting in
blur and noise, approaches have been developed to math-
ematically reverse the degrading effects by deconvolution
algorithms (30, 31). Most deconvolution algorithms require
for linear systems as the image of a point source of the
microscope (for a review see 32). For every study the
point spread function and the measured one has to be
compared (28). We have compared the two approaches in
this study and did not find a discernible difference (data not
function as a routine. Using a deconvolution algorithm the
relative spatial arrangement of fine particles, and even of
nanoparticles, within RBC was possible.
FIGURE 2. TEM images of different particles within RBC. The images show entering of 0.2 µm polystyrene particles (a; arrows), at higher
(c, c′, d; white arrows) 0.025 µm gold particles are taken up by RBC. The particles are not membrane-bound. Bigger agglomerates (>0.2
µm) are always observed outside the cells (d; black arrow).
43569ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 40, NO. 14, 2006
We have shown that nanoparticles enter RBC, but for
ultrastructural analysis TEM had to be applied. For this
analysis we could not use polystyrene particles, as we were
not able to detect polystyrene nanoparticles in RBC. There-
fore, we applied electron dense nanoparticles (gold) and
analyzed their intracellular localization with TEM. We were
able to show that gold particles were within the RBC and
tetroxide or uranyl acetate used for increasing the contrast,
conventional TEM is not adequate. Thus, analytical TEM,
which allows the visualization of the chemical composition,
was used to analyze the ultrastructural localization of gold
(made more easily detectable by silver enhancement) and
ultrafine TiO2 particles. It was previously shown that it is
possible to identify and localize particles of diameters less
than 0.1 µm in biological objects with EFTEM (26). In this
study we also applied analytical TEM since it is essential to
clearly identify nanoparticles. Other studies have shown
membrane-bound vacuoles containing mostly large ag-
gregates of TiO2 in A549 cells (33) or the uptake of ceria
nanoparticle agglomerates in human lung fibroblasts (34).
However, in these studies only conventional TEM was
employed to detect the particles, and it might be that single
nanoparticles could not be identified.
effects and pharmacokinetics of airborne particles in cell
see 35). Particles with diameters >1 µm usually remain on
the epithelial surface upon their deposition (9, 10, 17) and
are subject to clearance by cough, mucociliary transport,
nanoparticle types, particularly those of <0.01 µm size, that
they are transported across the olfactory epithelium and
nanoparticles into cells are still not yet known. None of the
endocytic pathways, which all include vesicle formation by
an actin-mediated process, are likely to account for the
translocation of nanoparticles (21). The translocation of
particles may also occur by unspecific means, including
diffusion, trans-membrane channels, and electrostatic, van
“adhesive interactions” in adhesion science and technology
(for a review see 40, 41).
FIGURE 3. EELS images of TiO2and silver enhanced gold particles. Ultrafine or small aggregates (<0.2 µm) of TiO2(a, white arrow) and
silver enhanced gold particles (c, white arrow) are found inside RBC and are never membrane-bound. The white open circles mark the
positions were the energy loss analysis was performed. The corresponding energy loss spectra (black lines) are shown as insets in (a)
and (c). The dotted lines show the background. Notice the structures that are similar to TiO2particles but that were identified to contain
no titanium (a, black arrow). Bigger agglomerates (b, d, white arrows) are found outside and sometimes attached to the cell surface. The
circles mark the region of the element analysis.
VOL. 40, NO. 14, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY94357
We have used isolated RBC, which are nonphagocytic
cells, to study the influence of size, charge, and material on
the entering process of nanoparticles into cells. The micro-
showed that very small fine particles e0.2 µm in diameter
and nanoparticles can enter RBC. The aggregation behavior
(34). In our experiments all types of particle suspensions
of the calculated point spread function and the majority of
the particles were seen as single particles. Sonification of
TiO2 particle suspension could not eliminate particle ag-
gregation totally. TiO2 nanoparticles were found as large
e0.2 µm in diameter. Larger aggregates were found to be
stuck at the surface membrane of the cells. Colloidal gold
particles do not tend to aggregate as much as TiO2nano-
particles, since their protein coating stabilizes the particles
and minimizes aggregation. For particle characterization
colloidal gold particles diluted in RPMI 1640 were adsorbed
on Formvar-coated copper grids. Gold particles were found
to be distributed randomly on the grid. In some cases small
clusters of 2 or 3 particles were found according to the
manufacturers specification (data not shown). Aggregates
larger than 0.2 µm were found to be attached to the cell
membrane, whereas smaller aggregates and single particles
were found within cells. Each experiment was repeated
several times. In addition different particle concentrations
and incubation times, as well as dilution of the noncharged
same result, i.e., the observation of single nanoparticles or
aggregates e0.2 µm within the cells (data not shown).
Fluorescently labeled noncharged, as well as negatively
charged, nanoparticles were counted and for both particle
types only 0.6-0.8 particles/cell were seen. This indicates
that there is no accumulation of nanoparticles in RBC.
We showed evidence that RBC contained nanoparticles
of various materials, but neither endocytosis which is based
on vesicle formation, nor any actin-based mechanisms, are
likely to account for nanoparticles’ translocation into the
cell (21). There may be no means on the cellular level to
prevent, influence, or direct their uptake. However, in the
past years, nanobiotechnology has been applied to improve
based probes for tumor diagnostics and therapeutics (42),
and it has been shown that for a strong affinity for target
one hand, the ability of free location and movement within
intracellular targets could result in enhanced therapeutic
efficacy and reduced toxicity.
Sophisticated microscopic methods have to be used for
the detection of nanoparticles within RBC. We did not see
any difference in particle entering with respect to differing
surface charges or surface chemistry, as we used three
different particle types, metal, metal oxide, and synthetic
polymeric materials uncharged or with negative or positive
charges. We may conclude from these results that particle
size is the most important factor for their translocation into
In future investigations we will apply the well-established
interactions in a more advanced cell culture modelsa triple
cell co-culture model of the human airway barrier. It has
been designed to simulate the cellular part of the epithelial
airway barrier of the respiratory tract represented by
macrophages, epithelial cells, and dendritic cells (44).
We are grateful to Barbara Tschirren and Sandra Frank for
the Swiss Agency for the Environment, Forests and Land-
scape, and the Silva Casa Foundation. The authors declare
that they have no competing financial interest.
EELS Electron energy-loss spectroscopy
EFTEM Energy-filtering transmission electron micros-
LSM Laser scanning microscopy
PBS Phosphate-buffered saline
Transmission electron microscopy
RBC Red blood cells
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Received for review November 10, 2005. Revised manuscript
received January 28, 2006. Accepted February 15, 2006.
VOL. 40, NO. 14, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY94359