Analyzing the Biological Entity of Nanomaterials - Safety of Nanomaterials along Their Lifecycle - page 63

referred to as the “Vroman effect” (Vroman et al. 1980). These dynamic changes in
the composition of the protein corona add another dimension to the already com-
plex situation. As nanomaterials can move through different physiological compart-
ments the protein composition of the corona will change and therefore it is important
to study the protein corona also over time. But importantly, the corona will never
be completely exchanged; traces of the “primary” corona will always be retained
such that the passage of a nanomaterial may be recapitulated via identification of
the adsorbed biomolecules. The protein corona thus serves as a “fingerprint” and
enables us to “read” the passage route of a nanomaterial.
Studying the protein corona completely, also over time, is highly important as
adsorbed proteins may shield toxic nanomaterial properties or vice versa increase
toxicity (i.e., by increasing cellular uptake), may trigger cellular effects (e.g.,
enhanced presentation of nanomaterials to cells via adsorbed proteins), or may cause
interference with cellular signaling pathways (Johnston et al. 2012). Furthermore,
protein adsorption is likely to lead to altered size distribution and state of dispersion
of nanomaterials (Dawson, Anguissola, and Lynch 2012; Lesniak et al. 2012). The
dispersion profiles of nanomaterials in relevant biological fluids (containing protein,
lipid, sugars, and ions) may differ significantly from profiles in other buffers, as bio-
molecules and ions can shield electrostatic interactions between nanomaterials, and
thereby lead to formation of agglomerates (Nichols et al. 2002; Maskos and Stauber
2011). Especially the state of dispersion is of tremendous importance to nanotoxi-
cological studies, as size of a nanomaterial is the key parameter that in most of the
cases inversely correlates with toxicity. Agglomerated nanomaterials may behave
in a completely different way in test assays compared to nanomaterials distributed
in the nanometer range, as agglomerated nanomaterials will sediment and are thus
most likely to be available for cells to a larger extent, which of course also influences
the toxicological endpoint (Dawson, Anguissola, and Lynch 2012).
A key step toward meaningful toxicity assessment of nanomaterials is therefore
sample preparation, which means dispersion of the nanomaterials into an aqueous
test medium. Some additives have been identified that are capable of stabilizing
nanomaterials in aqueous media, such as albumin or citric acid (Schulze et al. 2008;
Ramirez-Garcia et al. 2011). However, there is still a lack of harmonization of meth-
ods for dispersing nanomaterials, and profound differences can be observed depend-
ing on the composition of the dispersion medium, as well as dispersion procedure
(Dawson, Anguissola, and Lynch 2012). The major challenges in nanotoxicological
studies are still the selection and application of suitable methods to obtain meaning-
ful and conclusive data (see Section 4.1.3), as well as the choice of suitable biological
matrices representing the respective physiological compartment within the human
body (see Section 4.1.4).
4.1.3 m ethodologiCal a PProaChes for I n S Itu
C haraCterization of n anomaterials
Characterizing nanoparticles in a given biological environment and especially deter-
mining their biomolecule corona is an analytical challenge. One needs to decide on a
case-by-case basis which technique to use as there is no one-fits-all method available.