Analyzing the Biological Entity of Nanomaterials - Safety of Nanomaterials along Their Lifecycle - page 64

As there are excellent review articles available (Aggarwal et al. 2009; Mahmoudi
et al. 2011), we will give only a brief overview here. Basically, there are two general
approaches. It is possible to study the nanomaterial directly in the test conditions ( in
situ ). On the other hand, it is also possible to reisolate the nanomaterial including
adsorbed biomolecules for subsequent analysis ( ex situ ) (Walkey and Chan 2012;
Maskos and Stauber 2011). However, in the literature the term in situ characteriza-
tion of nanomaterials is often used for both in situ and ex situ approaches to study
nanomaterials under test conditions in contrast to nanomaterial characterization as-
synthesized , which is typically performed in water. As each method has limitations,
typically a combination of different methods ( in situ and ex situ ) is needed to fully
describe nanomaterials in complex (i.e., biological) matrices.
Suitable in situ methods are limited in number. It is possible to use methods
that are suitable to study size and size distribution such as dynamic light scattering
(DLS) or nanoparticle tracking analysis (NTA) to check for an increase in hydro-
dynamic radius due to formation of a protein corona (Montes-Burgos et al. 2010).
DLS uses coherent laser light and measures the angular dependent scattering occur-
ring from particles. The fluctuations over time will depend on particle movement,
which is dependent on particle size. NTA uses the same main principle but does not
measure the mean scattered light instead here each particle is followed individually
(referred to as “tracking” of particles). Both approaches will deliver information on
the size and size distribution and eventually also on the shape of the particles or on
interactions between them. Care needs to be taken as some matrices will interfere
with DLS or NTA (e.g., lipid vesicles or protein aggregates may interfere with DLS
or NTA). Similar approaches are analytical ultracentrifugation (AUC) or differ-
ential centrifugal sedimentation (DCS), where particles will sediment according
to size during centrifugation (Wohlleben 2012; Schulze, Rothen-Rutishauser, and
K reyl i ng 2011).
However, often the increase in hydrodynamic radius cannot simply be explained
by the formation of a protein corona as it is much more pronounced. Some nano-
materials will agglomerate in biological matrices. Thus, one needs to be careful in
interpreting the results from techniques that primarily determine particle size and
size distribution such as DLS, NTA, AUC, or DCS.
Other useful methods are spectroscopic methods such as fluorescence spectros-
copy, Raman microspectrometry, or circular dichroism (CD), which are especially
useful in detecting structural changes in interacting biomolecules (e.g., structural
changes in protein, partial denaturation). For instance, Raman microspectrometry
may be especially useful for metal nanoparticles (in particular silver or gold), which
lead to very strong Raman signals due to signal enhancement by several orders
of magnitude (so called surface-enhanced Raman scattering) because of surface
plasmons. This allows for studying protein corona in situ directly inside biological
matrices, or even inside cells (Drescher et al. 2013).
Fluorescence correlation spectroscopy (FCS) has been successfully applied to
study protein corona thickness and layer structure (Maffre et al. 2011; Milani et al.
2012). However this technique is limited to the study of the interactions of a single
protein and a nanoparticle. In addition, the nanoparticles need to be monodisperse
and with a narrow size distribution.