Lessons Learned from Pharmaceutical Nanomaterials - Safety of Nanomaterials along Their Lifecycle - page 117

precise information on chemical components. Due to these techniques it is possible
to determine impurities or reinforce the pureness of a sample. Another issue is con-
formation of the functional groups on the nanoparticle surface. The available groups
can be identified by various staining methods, some of them used since decades. The
Ellman's assay is capable of detecting sulfhydryl groups (Ellman 1959), trinitroben-
zenesulfonic acid (TNBS) assay can estimate chemical free amino groups (Habeeb
1966), and surface hydrophobicity can be at least in relative terms determined via
Rose Bengal adsorption (Müller 1991).
There is now a common understanding in nanomedicine that a thorough physi-
cochemical characterization is a prerequisite for medical application and that this
does include also the agglomeration/aggregation status of particles in biological
fluids.
6.3.2 P artiCle -C ell /t issue i nteraCtions
There are various tests which can be used to investigate the interaction of nanopar-
ticles with cells/tissues. These interactions are possible to be investigated at three
different levels. First and most common is to test cytotoxicity, which compares living
cells against dead cells and concludes cell viability. The immunostimulatory effects
and the capability to induce genetic mutation received less attention, which has to
be changed.
6.3.2.1 Cytotoxicity or Cell Viability
One of the first tests before using nanopharmaceuticals in clinical studies is to
investigate their impact on cell viability or proliferation in cell culture. For that
issue, colorimetric- and luminescence-based assays were developed to directly or
indirectly measure their cytotoxicity. Assays for different cytotoxicity mechanisms
are available and several should be used to verify the pharmaceutical usability of
nanoparticles (Weyermann, Lochmann, and Zimmer 2005). These assays are based
on association of cell death versus proliferation. One assay measures the activity
of enzymes that are available to metabolize a tetrazolium salt (MTT) to its water
insoluble formazan dye. Another assay enables visualization of cell membrane dam-
age, which indicates a loss of cell membrane integrity. By cell damage a cytoplasmic
enzyme, lactate dehydrogenase (LDH), is released into the extracellular supernatant.
In the cell culture supernatant LDH is still active and can be used in an enzymatic
assay. Just like the MTT assay a tetrazolium salt is reduced to a red formazan dye.
These are two colorimetric assays. One proves cell viability (MTT assay), whereas
the other proves the cell membrane damage therefore cell death. A further method
exploits the consumption of the “molecular fuel” adenosine-5-triphosphate (ATP).
In many cellular processes and metabolism ATP supplies the chemical energy. All
viable cells can be determined by a bioluminescent method, which utilizes a cata-
lytic enzyme (luciferase). Luciferase generates light by catalyzing ATP and the sub-
strate luciferin. This bioluminescent assay is able to measure metabolically active
cells (Crouch et al. 1993). In most cases, microtiter plates were used to have a high
throughput rate using 96-well plates. This miniaturization allows us to analyze a
high number of samples simultaneously.