Analyzing the Biological Entity of Nanomaterials - Safety of Nanomaterials along Their Lifecycle - page 65

Other in situ methods are surface plasmon resonance (SPR) and isothermal titra-
tion calorimetry (ITC), which both allow for determination of thermodynamic
characterization of the nanomaterial-biomolecule interaction. SPR is mainly used
for studying the adsorption kinetics (i.e., association and dissociation constants,
k on and k off ), whereas ITC enables us to determine the stoichiometry of the binding
(Mahmoudi et al. 2011). This technique is also limited to the study of individual
proteins only.
One major disadvantage of all these approaches mentioned here is the fact that
none will allow for identification of adsorbed biomolecules. Although they may
deliver one piece of information such as the presence of a corona, corona thick-
ness, or structural changes of adsorbed proteins, they will never allow mapping of
adsorbed components, which is possible only with ex situ approaches.
The most challenging step for ex situ approaches is the isolation of the nanoma-
terial-biomolecule complex, which should be performed ideally without perturba-
tion of the system (which is literally impossible to achieve). A common method to
separate nanomaterial-biomolecule complexes from unbound material is centrifu-
gation, followed by several washing steps (Aggarwal et al. 2009). Centrifugation,
especially in combination with several washing steps, will remove weakly bound
biomolecules from the nanomaterial surface. What is recovered after the subsequent
washings steps is therefore often referred to as the “hard corona” (Lundqvist et al.
2008), which includes molecular species bound with high affinities to the nanomate-
rial. Other approaches to separate nanoparticles from nonbound biomolecules are
magnetic separation, size exclusion chromatography (SEC), differential centrifu-
gation (DC), or field-flow-fractionation (FFF) (Aggarwal et al. 2009; Walkey and
Chan 2012; Maskos and Stauber 2011). Methods such as SEC or FFF may eventually
allow also detection of proteins belonging to the “soft corona,” which are less tightly
bound and are proteins of the corona that are prone to exchange processes over time
(Maskos and Stauber 2011; Cedervall et al. 2007).
Once the nanomaterial-biomolecule complex is isolated, the adsorbed biomol-
ecules may be analyzed directly on the surface, which is possible for instance when
studying lipids using secondary ion mass spectrometry (Grenha et al. 2008). Proteins
may be digested directly on the surface and then analyzed in gel-free approaches or
may be eluted by application of high temperature, detergents, high salt concentra-
tions, enzymatic digestion, or extraction with organic solvents (which is relevant
for lipid analysis), and then analyzed via gel electrophoretic approaches. However,
there have been cases reported where complete protein elution was not possible due
to strong interactions with the nanomaterial—a fact underlining the need to include
meaningful controls in experiments (Schulze, Rothen-Rutishauser, and Kreyling
2011). Following isolation and elution, the most common method for protein analy-
sis is poly(acrylamide) gel electrophoresis (PAGE), which can be performed in a
one- and two-dimensional manner. Especially one-dimensional (1D) SDS-PAGE is
often used, as it is relatively easy and affordable and can be combined subsequently
with other highly sensitive mass spectrometric methods (Lundqvist et al. 2008;
Hellstrand et al. 2009; Monopoli et al. 2011). Two-dimensional (2D) gel approaches
offer the advantage of separating the complexity of bound proteins very efficiently
and allow discrimination of protein species (e.g., different isoforms or different