Analyzing the Biological Entity of Nanomaterials - Safety of Nanomaterials along Their Lifecycle - page 71

serum is heat inactivated compared to nonheat-inactivated serum, which in turn also
influences the outcome of the toxicity study (Lesniak et al. 2010).
For a deeper molecular understanding of the actions taking place on the nanopar-
ticle surface, however, one even needs to reduce the complexity of the whole system
further down to a single protein-nanomaterial system. This allows us to study the
interaction in detail and for instance to follow changes occurring in protein structure
during interaction with nanoparticles. Therefore single nanoparticle systems prove to
be very useful for certain types of analysis as well.
4.2.3 s ingle P rotein s ystems
As the surface of the nanomaterials is more or less strongly curved depending on
nanomaterial size and shape most proteins will have to adapt their three-dimen-
sional (3D) structures at least partially to fit onto that surface. Other factors deter-
mining this interaction are charge-dependent interactions as the surface charge of
nanomaterials will attract oppositely charged amino acids and repel those of similar
charge. Hydrophobic interaction will also contribute to partial unfolding of proteins.
Eventually for some nanomaterials covalent bonds may also be formed for instance
with cysteine residues. Thus taken together, often protein 3D structures will change
during interaction with nanomaterials. As this has already been explained in detail
earlier in this chapter we will only summarize the most important aspects as they
are important to understand the studies, which will be discussed here. Interactions of
proteins with nanoparticle surfaces may lead to uncovering of so-called “cryptic epi-
topes,” which previously have been hidden inside the protein (Lynch, Dawson, and
Linse 2006; Deng et al. 2010). Partial unfolding will also influence protein-protein
interactions, eventually changing protein fibrillation or aggregation as it has been
shown for β2-microglobulin (Lynch, Dawson, and Linse 2006) or amyloid β protein
(Cabaleiro-Lago et al. 2008) interacting with polymeric nanoparticles. Of course
changing protein structure will also influence protein function, which has been ana-
lyzed for instance for chymotrypsin and peroxidase interacting with MWCNTs or
lysozyme interacting with ZnO (Wu, Zhang, and Yan 2009; Chakraborti et al. 2010).
Shang and coworkers could nicely show that the adsorption of BSA onto a gold
nanoparticle surface changes protein secondary and tertiary structures (Shang et al.
2007). They used fluorescence spectroscopy, FT-IR, and CD-spectroscopy to prove
that tryptophan residues moved into a different environment when BSA attached to
the nanoparticle surface and that the overall helicity of the protein changed, depend-
ing on the pH value of the environment.
Pan and coworkers applied quantitative stopped-flow measurements to unravel
the underlying kinetics of interaction of GB1 protein with latex nanoparticles to
understand precisely how this interaction is established, and they furthermore ana-
lyzed how changes in pH and ionic strength would influence this system (Pan et al.
2012). They could show that upon adsorption GB1 is partially unfolded, and they
could calculate the folding and unfolding rates of GB1 while in contact with the
nanoparticles. Interestingly both rates are much slower than those for free GB1 in
solution indicating that the free-energy barrier between folded and unfolded states
is increased. In addition, in solution the folded state is more stable than the unfolded