Analyzing the Biological Entity of Nanomaterials - Safety of Nanomaterials along Their Lifecycle - page 81

recovered from BAL. Analysis of adsorbed lipid components using LC-MS revealed
that mainly PCs and PGs (the most abundant species) had bound to the SWCNTs
and a complete coverage of the nanomaterial surface with PLs was estimated and
modeled. Furthermore, SWCNTs coated with surfactant PLs were studied in vitro in
a mouse macrophage model (RAW 264.7), both in the presence and absence of addi-
tional SP-D. Interestingly, lipid-coated SWCNTs demonstrated an increased uptake
by the macrophages, whereas this was slightly enhanced when studied in the pres-
ence of SP-D.
Ruge and coworkers used magnetite nanoparticles and obtained similar results
demonstrating that SP-A and SP-D can bind to such nanomaterials in material-
dependent manner. Again the adsorbed surfactant proteins were able to enhance
uptake of these particles by alveolar macrophages (Ruge et al. 2011; Ruge et al.
2012). The biological effects were mainly mediated via adsorbed proteins whereas
protein binding was dependent on the material surface properties. This was shown
by testing different types of magnetite particles, some of which were more hydropho-
bic and others were more hydrophilic. Interestingly, when preincubating the nano-
materials with complete native surfactant, the lipids would modulate the effects seen
before, now resulting in very comparable results for the different nanomaterials. This
indicates that interactions between surfactant proteins and lipids may actually coun-
terbalance each other (Phelps 2001).
All these examples demonstrate that future studies are needed to understand the
processes of nanomaterial interacting with the lung lining fluid and cells. In other
words, bio-nano interactions in the lungs also are just as complex and as dynamic as
in the bloodstream. In both scenarios we may observe exchange processes leading to
an “evolution” of the corona but also synergistic effects may occur. PS is a lipid-pro-
tein mixture with a unique and complex 3D structure that originates from the inter-
play of its components (Serrano and Pérez-Gil 2006). Looking at these protein-lipid
interactions, it becomes clear that nanomaterial-adsorbed molecules will also interact
with nonbound molecules (which might be even part of surfactant membranes), add-
ing another level of complexity to the situation. Other than in the bloodstream where
the formation of the protein corona could be proven as an existing phenomenon, a
detailed identification of a “pulmonary corona” mapping all protein and lipid com-
pounds is still a missing milestone regarding the study of nanomaterials in the lungs,
and will be the subject of future investigations.
4.3.4 l essons l earned from n ano gem
Regarding the in situ characterization of nanomaterials after contacting lung lining
fluid, the availability of a suitable lung lining fluid model was one of the key issues
in the nanoGEM project. To investigate interactions between nanomaterials and sur-
factant phospholipids, commercially available Curosurf ® was chosen as a model for
the lipophilic fraction of PS. However, to study the binding of surfactant associated
proteins to these nanomaterials, this model is less suitable, as the majority of relevant
proteins is removed from this biological matrix during the lipophilic extraction of
Curosurf ® . To overcome this limitation, whole native surfactant isolated from por-
cine lungs, containing the relevant SP-A, B, and C, while only traces of SP-D can be