Analyzing the Biological Entity of Nanomaterials - Safety of Nanomaterials along Their Lifecycle - page 73

was shown that the total bound protein amount for negatively charged nanoparticles
(carboxyl-modified polystyrene nanoparticles) initially increased upon increasing the
amount of plasma added to a certain amount of nanoparticles. But then it decreased
and only increased again at much higher plasma amounts. In contrast, for positively
charged nanoparticles (amine-modified polystyrene) this was not observed. Here
there was a steady increase of bound protein upon increasing the amount of serum.
This may be explained by the fact that most of the serum proteins are negatively
charged under neutral pH conditions, and in good correlation with this the authors
could furthermore show that the composition of the coronas was different for nega-
tively and positively charged nanoparticles.
Finally, there is also correlation between the total protein adsorbed and surface
hydrophobicity of nanomaterials (Gessner et al. 2002). Gessner and coworkers
showed that with decreasing hydrophobicity the total amount of bound protein would
decrease and in addition this also altered the corona in a qualitative manner.
Taken together all these studies nicely show that the most important nanoparticle
characteristics influencing the protein adsorption and corona formation in a quan-
titative and qualitative way are size and shape of nanoparticles as well as surface
properties such as charge and hydrophobicity.
4.2.5 l essons l earned from n ano gem
Although several studies have already addressed the size-dependent effects of nano-
material interaction with serum proteins, much less is known about the other proper-
ties previously mentioned such as charge or hydrophobicity. Most of the knowledge
of how charge or hydrophobicity influences the interactions with serum proteins is
derived from quite ideal polymer type nanoparticles such as polystyrene or latex.
When nanoGEM was started, basically no study had addressed the influence of sur-
face functionalization on protein interaction for industrially relevant nanoparticles.
This gap of knowledge was identified as one of the major research topics for the
nanoGEM project. Industrially relevant nanoparticles were systematically varied
on the surface using different types of functionalization. For instance, nanoscaled
silicon dioxide (SiO 2 ) was applied in four different variants: without any specific
surface functionalization (SiO 2 naked), with an overall positive charge at neutral pH
(SiO 2 amino), with an overall negative charge (SiO 2 phosphate), and covered with a
short PEG chain (SiO 2 PEG).
The interaction with serum proteins was studied in cell culture medium contain-
ing 10% fetal calf serum (DMEM with 10% FCS) with 1D SDS PAGE and with
2D gel electrophoresis, in combination with mass spectrometry. Proteins bound on
particle surface were eluted, separated with 1D SDS PAGE, stained with Coomassie
dye, and assessed by densitometry. Taking into account normalization via five differ-
ent marker bands, the total amount of bound proteins could then be estimated. With
that approach the time-dependent formation of protein corona had been analyzed
(Figure 4.2a).
It becomes obvious that naked SiO 2 binds high amounts of proteins after 1 h,
whereas PEGylation of nanoparticle surface repels proteins, at least initially during
the first 2 h. However, as only 40% of the surface is covered with PEG and the PEG