Toxicological Effects of Metal Oxide Nanomaterials - Safety of Nanomaterials along Their Lifecycle - page 170

particle surface area dose, specific surface activity, and the proinflammatory effects
in vivo and in vitro . Their study also demonstrated the utility of in vitro assays for
predicting the ability of nanoparticles to cause inflammation in vivo on the basis
of their surface area and reactivity (Duffin et al. 2007). In addition, a concordance
between surface area dose of low-solubility, low-toxicity particles (LSLTP) that pro-
duce inflammation in vivo and the surface area dose of LSLTP that produce an IL-8
release in A549 cells in vitro was observed (Donaldson et al. 2008). These studies
suggest in vitro studies as a valuable complement to animal studies.
In line with the results from assessing ROS formation and decrease in cell viabil-
it y, TiO 2 nanoparticles displayed an overall greater immunotoxicity in A549 cells
when compared to rutile TiO 2 nanoparticles demonstrating the importance of crys-
talline structure in nanoparticle-induced inflammatory responses. In particular, the
release of IL-8 observed in human dermal fibroblasts and A549 cells after exposure
to anatase TiO 2 nanoparticles was found to be significantly lower when the cells were
exposed to rutile TiO 2 nanoparticles (Sayes et al. 2006).
8.3 EFFECTS OF METAL OXIDE NANOPARTICLES
ON PRIMARY ALVEOLAR MACROPHAGES
Many types of nanoparticles form agglomerates inside the lung alveoli (Gosens
et al. 2010; Morfeld et al. 2012), where they are ingested by alveolar macrophages.
Measuring the in vitro cytotoxicity with freshly isolated primary alveolar macro-
phages (AM) therefore gives first insight into whether a particle is cytotoxic to this
particular cell type, which is chiefly responsible for clearing the lung from particle
loads. Furthermore, a particle-induced release of ROS or of proinflammatory cyto-
kines (such as TNFα) from AM can be analyzed. This yields plausible information
to what extent cells of the lung epithelium may be indirectly compromised by parti-
cle-loaded AM. However, to produce meaningful in vitro results, which may be even
predictive for in vivo findings, several requirements have to be met, two of which
appear to be of major importance: first, the mean particle load of AM in vitro should
be adapted to what is obtained during an inhalation experiment. As AM in the lung
actively collects particles, surprisingly high mass per cell values are reached and up
to 90 pg/cell have been found secondary to inhalation exposure to AlOOH nanopar-
ticles (Pauluhn 2009). Therefore, in vitro tests are carried out with fixed numbers
of AM per culture well in order to achieve a defined loading with particles of up
to 120 pg/cell after 16 h (Rehn, Rehn, and Bruch 1999; Bruch et al. 2004). Second,
in vitro tests with AM should be carried out in the absence of proteins, as inhaled
particles are primarily not covered by proteins. Furthermore, serum-free conditions
allow us to study the primary effects of particles on AM.
In the nanoGEM project, the biological effects of different surface coatings were
investigated for ZrO 2 nanoparticles, which are widely used as a ceramic filler mate-
rial. ZrO 2 nanoparticles are insoluble, and exhibit no measurable ROS generation
at their surface (in contrast to, e.g., TiO 2 nanoparticles), which predestines them for
investigations on the biological effects of surface modifications. In particular, 10 nm
ZrO 2 nanoparticles were modified on their surface either with a caustic polyacrylate
(ZrO 2 .acryl), a polyoxocarbonic acid (ZrO 2 .TODS), an amino silane (ZrO 2 .a m ino),