Analyzing the Biological Entity of Nanomaterials - Safety of Nanomaterials along Their Lifecycle - page 79

that they lack important components (e.g., SP-A and SP-D), and therefore do not
completely reflect the physiological situation. Moreover, depending on the extrac-
tion method and origin (i.e., species), they also differ in PL composition. Hence, the
most physiological model for PS is still purified native surfactant, which should be
the model of choice when nanomaterials are characterized under conditions close
to the in vivo situation. Nevertheless, there are some methodological pitfalls to con-
sider when working with this model. The disruption of the interfacial surfactant
film during isolation and purification leads to formation of vesicular structures in an
aqueous environment. These large multilamellar vesicles (also referred to as “large
lamellar bodies”) are prone to sedimentation during centrifugation when nanomate-
rial-biomolecule complexes are separated. Unbound surfactant material can thereby
cosediment with the nanomaterials, leading to false positive results and actually pro-
duce a very high background, which strongly interferes with most types of analysis.
Furthermore, in situ characterization of nanomaterial sizes and size distribution in
purified native surfactant is hard to accomplish, as basically for the same reason the
large lamellar bodies interfere with the assays such as light scattering.
4.3.3 l essons l earned from P uBlished s tudies
Pulmonary toxicity assessment of nanomaterials currently is focused very much on
two aspects. First, biophysical investigations are performed to study the effect of
nanomaterials on the functionality of PS to maintain respiratory mechanics. Second,
the interaction between nanomaterials and components of the lung lining fluid is
probed to study binding phenomena, and how adsorbed biomolecules trigger the
biological response of the lungs toward such materials. Biophysical investigations
are generally performed by mixing the nanomaterial and surfactant material in an
organic phase, followed by injection of this solution onto an aqueous phase. As the
solvent quickly evaporates, the remaining interfacial film (including the nanomate-
rial) can be compressed using a film balance (e.g., Langmuir-Blodgett film balance)
to generate surfactant-like multilamellar structures. One measures the compress-
ibility during this process, which is one parameter indicating the functionality of the
surface film. After compression, such films can be transferred to mica substrates and
imaged using atomic force microscopy (AFM). Several such studies have been per-
formed in the last few years. They use either artificial surfactant models consisting
of single PLs, eventually combined with purified SP-B and/or SP-C (Harishchandra,
Saleem, and Galla 2010; Sachan et al. 2012), or lipophilic extracts such as Curosurf ®
or Survanta ® (Schleh et al. 2009; Tatur and Badia 2012). Interestingly, most of the
studies could demonstrate a concentration-dependent adverse effect of the respective
tested nanomaterial on the biophysical function of the surfactant film. Moreover,
a study by Fan and coworkers demonstrated a time-dependent adverse effect of
hydroxyapatite nanoparticles on the biophysical function of PS (Infasurf) (Fan et al.
2011). However, when cellular toxicity of these nanomaterials was assessed in a
human bronchial epithelial cell line, no toxic effects could be observed. Actually,
insertion of nanomaterials into the surfactant layer seemed to induce structural defi-
ciencies in close proximity to the nanomaterial, and partly even agglomeration of
surfactant components was observed. Although further consequences of such effects