Analyzing the Biological Entity of Nanomaterials - Safety of Nanomaterials along Their Lifecycle - page 82

detected using 2D PAGE and mass spectrometry (unpublished nanoGEM data), was
chosen to mimic the lung lining fluid.
Isolations were thoroughly characterized regarding their lipid as well as protein
composition. Comparisons of the molar compositions of major lipid species (PC,
PG, PI, and phosphatidylethanolamine) were in good correlation with values for PS
described in the literature. However, isolated native surfactant forms large lamellar
structures when dispersed in aqueous media, which furthermore tends to precipitate
during centrifugation. Due to the physicochemical properties of the nanomaterials
studied in nanoGEM, however, centrifugation was a necessary technique to sepa-
rate nanomaterials from unbound biomolecules after dispersion in biological fluid.
To exclude cosedimentation of the nanomaterials under investigation and unbound
vesicular structures from native surfactant (possibly leading to misinterpreted
adsorption results), a modified dispersion protocol was established. Particles were
dispersed for 1 h in deuterated water (D 2 O) at 37°C, which led to profoundly reduced
amounts of sedimented surfactant material after centrifugation. After incubation in
native surfactant, the binding of SP-A (as the most abundant surfactant protein) to
various nanoGEM particles surfactant was studied using this improved dispersion
protocol. By means of 1D SDS-PAGE with subsequent image analysis of the SP-A
bands (~34 kDa), the amount of adsorbed protein could be determined. The data
depicted in Figure 4.3 reveals an overall higher extent of SP-A binding for bare and
amino-modified SiO 2 nanoparticles, whereas other modifications such as SiO 2 PEG
or SiO 2 phosphate showed a rather moderate SP-A interaction.
On the one hand, such differences might be due to different surface proper-
ties (as for instance seen when comparing SiO 2 naked vs. SiO 2 PEG; compare also
Section 5.2.3). On the other hand, due to the broad variety of tested materials and
profound differences regarding their physicochemical properties (size, shape, sur-
face area, etc.), a direct comparison is only possible within narrow limitations.
Nevertheless, these data indicate that distinct variations can be observed among

16

14
12
10
8
6
4
2
0

nS control SiO 2

bare

SiO 2
amino

SiO 2
PEG

SiO 2
phosphate

FIGURE 4.3 Adsorption of SP-A to four different variants of SiO 2 . Nanoparticles were dis-
persed in native surfactant in deuterated water at 37°C for 1 h. After centrifugation, particles
were washed once and subsequently analyzed using 1D gel electrophoresis. The SP-A band
(34 kDa) was semiquantified using densitometry, and mean mass expressed in µg of adsorbed
SP-A ( n = 2).