Measurement of Nanoparticle Uptake by Alveolar Macrophages - Safety of Nanomaterials along Their Lifecycle - page 136

7.1 INTRODUCTION
The lung is the major entrance route for air borne nanoparticles (NP) into the body,
some of which have been shown to have considerable biologic effects (Oberdörster
et al. 2005a; Xia et al. 2009). Consequently, inhalation experiments appear best
suited to the toxicological testing of NP, since particles are distributed to all parts of
the lung in a life-like manner (Oberdörster et al. 2005b). However, the distribution
of nanoparticles in the lungs does not remain homogeneous. Several studies have
shown—and were also confirmed in the course of the NanoGEM project—that the
vast majority of the nanomaterial reaching the alveolar region appears in alveolar
macrophages (AM) (Gosens et al. 2009). The underlying mechanisms may involve
agglomeration of NP due to an interaction with components of the lung surfactant or
the lung lining fluid or pathways through the epithelium and/or interstitium (Rothen-
Rütishauser et al. 2007; Semmler-Behnke et al. 2007). In any case, particle laden
AM may reach the mucociliary escalator or travel toward mediastinal lymph nodes,
if their motility is not disturbed by phagocytized particles (Oberdörster et al. 1992).
However, AM will ingest particles also under cell culture conditions and it has been
shown that well designed in vitro experiments with AM are of some predictive
value with respect to the lung toxicity of micron-sized as well as nanosized particles
(Lanone et al. 2009; Rehn et al. 1999; Wiemann and Bruch 2009). The cellular
particle burden of AM may be adapted to the situation in vivo where a mean dose
of 60-90 pg/AM resulted from a 4 week inhalation exposure to 30 mg/m 3 (Pauluhn
2009). In this chapter, a light microscopic method is described with the help of which
the exposure of macrophages to (nano)particles as well as their uptake can be quanti-
fied in vitro. The topic of in vitro particokinetics becomes increasingly important for
correctly describing the cellular dose of cells exposed to nanoparticles (Teeguarden
et al. 2007). Particles with a hydrodynamic diameter larger than ca. 300 nm are
subject to instantaneous gravitational settling, whereas smaller nonagglomer-
ated particles stay suspended. Based on the mathematical description of diffusion,
agglomeration, and sedimentation the ISDD model was proposed which allowed
calculating as to whether a particle might sediment, which eases its contribution to
the cellular dose (Hinderliter et al. 2010). Data requirements for the model were not
only temperature, viscosity, particle size, and density but, in the case of agglomerate
formation, also the fractal dimension and packing factors. Importantly, it was shown
that the “difference between equivalent nominal media concentration exposures on a
µg/mL basis and target cell doses on a particle surface area or number basis can be
as high as three to six orders of magnitude.” Consequently, researchers have to be
aware that there will be a strong albeit indirect impact of particle properties on dose
rate, that is, the increment of cellular particle burden over time.
In this chapter, we will introduce an alternative approach to describe gravita-
tional settling of nanoparticles during in vitro testing. The method primarily takes
advantage of phase contrast micrographs serially collected with a time-lapse imag-
ing device operated under cell culture conditions. Agglomerated particles can be
viewed and counted in the lowermost optical plane in the absence or presence of
cells. Due to optical resolution the method is confined to agglomerates large enough
to be viewed with phase contrast optics (>0.3 µm) and has to be supplemented by