Analyzing the Biological Entity of Nanomaterials - Safety of Nanomaterials along Their Lifecycle - page 68

the use of fluids purified from biological systems such as the use of purified lung lin-
ing fluid. Buffers such as phosphate buffered saline (PBS) are a popular test medium
for nanomaterial characterization, which are also often used in the preparation of
nanomaterial for application (e.g., for i.v. injection or for instillation). In addition,
some artificial fluids are defined in the literature (i.e., resembling gastric fluids or
lysosomal fluid), some of which even may contain certain enzymes, and they have
been shown to be a very useful tool for characterizing nanomaterials. Still, such sys-
tems do not show the same complexity seen in in vivo body fluids as they typically
lack most of the proteins or other biomolecules.
The “gold standards” are such biological fluids that originate from the respec-
tive physiological compartment with which a nanomaterial can potentially inter-
act. With respect to the bloodstream, the relevant biological fluid (i.e., plasma) is
comparably easily available and can be used for in vitro studies on nanomaterial
biomolecule corona formation or for analyzing dispersibility. Lung lining fluid can
be purified from several sources—the most popular sources are rat or pig. However
lung lining fluid is more complicated to obtain (most often via lung lavage), and
is also difficult to standardize. For GIT typically only artificial fluids mentioned
previously exist. Before we go into detail, now we would like to spend a few words
on in vitro studies.
In vitro studies are typically performed with cell culture medium containing
serum. Serum content can vary from 1% to 20% and may vary in origin. Most often,
fetal calf serum is used but it is also possible to use horse, rat, human, or any other
serum. In recent times there are discussions going on to improve in vitro systems
in a sense that for rat cell lines, rat serum should be used; although for human cells
one should use human serum, and so forth. Hitherto, most laboratories still use long
established protocols using fetal calf or bovine serum. Serum may be used with or
without heat inactivation for in vitro studies. However, one should note that the com-
position of the cell culture medium—especially the selection and treatment of the
serum component (i.e., species, heat inactivation, concentration, etc.)—can interfere
or bias an assay, for which reason various control experiments need to be performed
(Lesniak et al. 2010).
In vitro studies using submersed cell culture systems may be very useful for
studying interaction of nanomaterials with cells from the blood or vascular system,
and for this purpose the in vitro system mimics the in vivo situation quite closely.
However, for other physiological compartments, cell culture media generally lack
specific components. So studies performed with submersed cell cultures using serum
additives will not be able to truly mimic oral uptake or inhalation. It may be possible
to improve such systems by first covering the nanomaterial for instance with lung
lining fluid in the case of analyzing inhalation before their addition to cell culture
systems containing lung cells. Another way would be to spike the cell culture media
with additives that are specifically relevant for the respective biological fluid, for
example, surfactant lipids, proteins, or mucins to mimic the lung lining fluid, or
saliva components and digestive enzymes in the case of the gastrointestinal fluid
(Lazzari et al. 2012). This may help to improve the in vitro systems in some cases,
but one needs to be aware of such differences when interpreting and comparing
results from different studies.