Analyzing the Biological Entity of Nanomaterials - Safety of Nanomaterials along Their Lifecycle - page 70

Taking the high complexity of plasma proteins into account, there is a huge vari-
ety of possibilities for forming a nanoparticle-protein corona. As explained in detail
in the introduction, the main nanoparticle properties affecting the composition of
the corona seem to be nanoparticle size and shape (affecting the curvature of the
nanoparticles) and surface properties such as charge or hydrophobicity (Lundqvist
et al. 2008; Tenzer et al. 2011; Gessner et al. 2000).
4.2.2 a vailaBle m odels and l essons l earned from P uBlished s tudies
The physiologically most relevant system to study would be human plasma or human
serum (e.g., plasma devoid of coagulation factors), which is easily available from
donations. In fact this system has been used in several studies so far (Tenzer et al.
2011; Lundqvist et al. 2008; Monopoli et al. 2011). Serum may be used either in a
pure state or further diluted. For instance, Monopoli and coworkers used 3%-80%
plasma for their study. PBS diluted plasma is often easier to analyze as it is less com-
plex, allowing in addition to protein corona analysis the study of other in situ charac-
teristics such as nanoparticle agglomeration, which with many techniques might not
be feasible in concentrated plasma. Other studies use bovine or fetal serum diluted in
cell culture medium, typically to a final concentration of 10%, which is the relevant
medium for in vitro experiments (Maiorano et al. 2010).
Therefore the choice of which medium to use for analysis strongly depends on
the questions underlying the study. For correlating results obtained from in vitro or
in vivo animal testing to humans or for directly assessing nanoparticle behavior in
human bodies, one certainly should aim to study nanoparticles in human plasma as
well. Although for interpreting results obtained from in vitro studies, cell culture
medium containing 10% serum is the medium of first choice for in situ characteriza-
tion of nanoparticles. Diluted serum can be regarded as a medium complex simulant
fluid, somehow mimicking pure serum. However one should keep in mind that some
major differences remain. The first major issue is the difference between serum, and
plasma as serum does not contain any coagulation factors that are present in plasma.
The second major issue is derived from dilution, although for reducing complex-
ity dilution may be a necessary step. The protein content in cell culture medium
containing only 10% serum is around 3 mg/ml and thus is significantly lower than
the protein content observed in plasma in vivo . Different percentages of serum may
yield completely different protein coronas as it has been shown already for silica or
polystyrene nanoparticles in a study by Monopoli and coworkers, with varied serum
content from 3% to 80% (Monopoli et al. 2011). But being the relevant medium for
in vitro cell culture containing all relevant serum proteins, cell culture medium con-
taining 5%-10% serum is a very good starting point for in situ characterization of
nanoparticles and for characterizing bio-nano interactions.
Apart from the question whether the serum should be used undiluted or diluted
another aspect is even more important to consider, which is heat inactivation of
serum. Typically or traditionally serum is heat inactivated for use in cell culture
but actually this will certainly alter the protein conformation. Nonheat-inactivated
serum resembles the in vivo situation more closely and most cell lines do not require
the serum to be inactivated anyhow. Protein corona will drastically change when