Measurement of Nanoparticle Uptake by Alveolar Macrophages - Safety of Nanomaterials along Their Lifecycle - page 152

becomes clear from particle number ( N ) that for a concentration of 11.25 µg/mL
and 22.5 µg/mL the quantity of AlOOH and CeO 2 particles inside the active
ROI (macrophage area) amounted to ca. 70% the control ROI. Consequently,
30% of both types of particles had been ingested. Importantly, no difference
between AlOOH and CeO 2 particles could be discerned with respect to phagocy-
tosis behavior at low concentration. At 45 and 90 µg/mL the sedimentation rate
( q ) of AlOOH increased whereas the relative amount of phagocytized particles
decreased to 22% (45 µg/mL) and 15% (90 µg/mL), indicating that the uptake of
particle could no longer keep pace with particle sedimentation. Interestingly, the
sedimentation rate observed for high concentrations of CeO 2 (45 and 90 µg/mL)
was markedly higher compared to low concentrations, most likely reflecting a
pronounced agglomeration. Consequently, the sedimentation process was finished
after 80-90 min. Another consequence of pronounced agglomeration and parti-
cle overlap was that the quantity of small single particles decreased. It was an
intriguing finding that the particle number in active ROIs increased. However, as
evidenced directly from the micrographs, macrophages continued to engulf pref-
erentially large or overlapping particles. This effect counteracted the agglomera-
tion in active ROIs and led to the seemingly paradox finding of increased particle
numbers. This example shows that the careful evaluation of phagocytosis data
should consider effects of agglomeration and needs to be confirmed by a direct
interpretation of micrographs.
7.4 DISCUSSION AND PERSPECTIVES
In this study, particle phagocytosis by macrophages was quantified from phase con-
trast images, taken from the lowermost optical plane, within which agglomerated
particles assemble by gravitational settling. The appearance of particle agglomer-
ates between macrophages is a prerequisite for their uptake and determines the dose
rate of an in vitro experiment. Unlike alternative approaches, which, for example,
try to describe this sedimentation process indirectly on the basis of particle charac-
teristics (Hinderliter et al. 2010), we propose an image based and, therefore, more
direct method to quantify particle sedimentation. To this aim, we developed a spe-
cial program to meet the most necessary requirements. The program development
was based on modeled or simulated data as well as on the experiences obtained
with real measurements. The program termed “AM Tracking” is able to automati-
cally detect the border of a macrophage in a complex surrounding, to track the
cells through the series of time-lapse images, and finally to count the number of
ingested particles. This allows us to directly describe the dose rate for single cells
and to study the impact of particle uptake on macrophage motility, which is of major
importance for lung clearance processes. Furthermore, the sedimentation of par-
ticles can be described by a sedimentation model, which is part of the program and
which is especially useful if dose rates of particles applied to cell cultures shall be
investigated.
The accuracy of the measurements is, of course, limited by many factors and
has not yet been confirmed by other methods such as the quantification of particles
in single cells, for example, by mass spectroscopy methods. However, first results