Analyzing the Biological Entity of Nanomaterials - Safety of Nanomaterials along Their Lifecycle - page 75

overlap in the protein corona of SiO 2 naked and SiO 2 PEG after 24 h incubation
time. As explained before, this may be due to the incomplete coverage of the surface
with PEG and due to the short PEG chain length. Interestingly there is only little
overlap between positively charged SiO 2 (SiO 2 amino) and negatively charged SiO 2
(SiO 2 phosphate). SiO 2 amino binds more total proteins compared to SiO 2 phosphate
in the first 2 h, and the total bound protein amount stays rather constant over 24 h
whereas for SiO 2 phosphate it steadily increases. Eventually this may be explained
by the fact that most serum proteins are negatively charged at neutral pH and thus are
more likely to interact with positively charged nanoparticles. Taken together also for
industrially relevant nanoparticles such as SiO 2 , the surface properties strongly influ-
ence the proteins attaching to it, both in a quantitative and a qualitative way. Clearly
material characterization as-synthesized and material characterization in situ need
to be combined to obtain meaningful results that later on can be correlated to the out-
come of toxicity testing for deriving (quantitative) structure-activity relationships.
4.2.6 C onClusions and C onsiderations for f uture s tudies
Keeping in mind how important the protein corona is for the fate of the nanoparticle,
influencing cellular uptake and toxicity, one should consider presenting nanopar-
ticles to the test system in a physiologically relevant manner being as close as pos-
sible to the in vivo situation. Although most cell culture protocols use fetal calf or
bovine serum as this is easy to obtain and rather cheap, one should surely consider
using human serum for culturing human cell lines and therefore also for in situ
characterization of the bio-nano interface. The use of serum from a different species
will introduce another variable into the system. There is a possibility that some of the
adverse effects seen in in vitro studies may be caused by the introduction of foreign
protein species into the cell. In addition, in all cases serum should not be heat inacti-
vated as this will change the protein structure with a strong influence on the protein
corona as well as on the outcome of the toxicity studies.
Batch-to-batch variations occurring for different serum batches are another aspect
that deserves attention. To solve this issue, one may pool several serum batches,
sometimes pooled serum is also commercially available (e.g., for cell culture pur-
poses), or analyze several independent serum batches. One other major experimental
problem is related to background, which arises from different reasons. Firstly, espe-
cially the highly abundant proteins will bind to any surface be it a nanoparticle or a
test tube. Therefore, care needs to be taken when choosing plastics or glassware used
for the studies. Proteins, especially highly abundant ones such as albumin, may also
be trapped when pelleting nanoparticles within the nanoparticle pellet and might
not be efficiently removed in washing steps in particular when nanoparticles under
study are not ideal and are prone to aggregation. Of course extensive washing and
centrifugation steps will certainly help to improve or overcome this issue, but finally
one may only analyze the so-called “hard” corona via this approach. This is why
most studies focus on “hard” corona analysis and typically include a series of wash-
ing steps until the washing solution is devoid of protein.
In addition, serum proteins are prone to aggregation, especially when the serum
has been frozen. Such serum aggregates may even be visualized with DLS or