Analyzing the Biological Entity of Nanomaterials - Safety of Nanomaterials along Their Lifecycle - page 59

of a so-called corona), and partial dissolution (i.e., release of ionic species). This
clearly means that the nanomaterial that finally reaches the cell surface will have
interacted, for example, with serum-containing cell culture medium in the case of
in vitro test conditions or with lung lining fluid, gastric juices, or other body fluids
under in vivo conditions, and certainly the characteristics of the material will not be
of the same type as it has been produced, and extensively characterized. Basically,
the same holds true for any nanomaterial that is formulated into a final product,
where also many changes will occur, which however will not be covered within this
chapter. Most importantly, if changes in physicochemical properties of a nanomate-
rial in situ are not taken into consideration, one may actually misinterpret results
from toxicity testing.
To overcome this problem, Sayes and Warheit suggest a three-phase approach
for nanoparticle characterization with respect to nanotoxicological studies (Sayes
and Warheit 2009). Phase 1 covers the powder state and includes the assessment of
chemical composition, size, size distribution, surface area, and morphology. Phase 2
covers the characterization of the dispersions and includes tests on agglomeration
or aggregation, as well as the formation of reactive species. Phase 3 then includes
the nano-bio interface. Basically, it may be concluded that a nanomaterial needs to
be characterized at all stages of its lifecycle starting with the synthesis, followed by
particle characterization in aqueous media and then after coming into contact with
biological matrices and finally even inside cells or tissues. Typically, it will be eas-
ily feasible to study nanomaterials in simulated biological fluids as we will explain
later on in this chapter in detail. In contrast, characterization inside cells or in vivo is
extremely difficult to achieve due to technical limitations. Therefore, characteriza-
tion truly in situ , inside cells or tissues, is currently out of scope for most studies, but
might become feasible in a few years.
Direct characterization of nanoparticles in situ , for example, directly in the test
medium or simulated biological fluids has been extensively described in several pub-
lications (Sayes et al. 2008; Powers et al. 2007; Montes-Burgos et al. 2010; Maskos
and Stauber 2011). This is referred to as in situ characterization in contrast to the
characterization “ as produced ” or “ as synthesized ,” although some of the techniques
applied for so-called in situ characterization are not truly in situ but ex situ as we will
explain in detail later on. To summarize, nanomaterial characterization may not only
be viewed from a materials science perspective but nanomaterials in a biological
environment do actually represent a biological entity, which consists of the nanoma-
terial with the adsorbed layers of biomolecules. This is a highly dynamic complex
that will change over time.
Changes observed when introducing a nanomaterial from a pure abiotic into a bio-
logical environment are interconnected and dependent on each other. For instance,
if a nanomaterial strongly interacts with proteins tightly covering its surface, this
often leads to an increased particle agglomeration. On the opposite, particles that are
repellent to proteins might stay more stably dispersed. All these interactions with
the matrix or medium components not only change the physicochemical properties
such as size or zeta-potential but also strongly influence the interactions with liv-
ing systems and thus also the outcome of toxicity tests. Thus, these in situ proper-
ties need to be determined not only for a full characterization but also regarding