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8.4.1 Cytotoxicity and Cell Viability Assays
Cytotoxicity and cell viability assays are the most common studies to assess the
toxicological profile for a given compound or xenobiotic. These are important in the
definition of dose–response relationship and identification of sub-lethal concentrations for mechanistic studies. Definition of dose–response curves is fundamental for
extrapolating relevant toxicological parameters, such as minimum effective concentration (MEC), half-maximal effective concentration (EC50), and maximum effect
(E max ). These parameters can be used to compare toxicological profiles of different
formulations, as well as to investigate the contribution of constitutive components
in modulating nano-based formulation toxicity (Gunsolus and Haynes 2016).
Cytotoxicity assays measure cell death after a treatment with a cytotoxic drug or
compound, and they are generally related with cell death mechanisms such as
necrosis and apoptosis (i.e., programmed and accidental cell death). These mechanisms reflect the ability of a drug molecule to trigger intracellular suicide mechanisms or destroy cells. Not very different than any other compounds or materials,
NM-induced cell death assessment focuses on examining the mitochondrial membrane integrity or potential by the assessment of the apoptotic protein levels or DNA
fragmentation (Akhtar et al. 2014). While apoptotic proteins activation are apoptosis indicators, cell membrane damage is usually considered a necrosis marker (Love
et al. 2012).
Membrane damage can be determined by measuring either the release of cytosolic molecules as lactate dehydrogenase (LDH) enzyme or the entrance or uptake
of exterior molecules that can function as probes, such as trypan blue, propidium
iodide, or neutral red dye. The LDH assay measures the activity of the enzyme
released in cell culture medium after contact with the tested NM (Jing et al. 2015).
The released LDH converts pyruvate into lactate with the consequent reduction of
NADH into NAD+. Therefore, a decrease of NADH absorption peak in extracellular medium corresponds to an increase of extracellular LDH concentration
(Caballero-Diaz and Cases 2016). Extracellular LDH content can usually be quantified by colorimetric methods based on the conversion of tetrazolium salt into soluble dyed formazan, which are also available in commercial kits (Love et al. 2012).
As previously mentioned, trypan blue and propidium iodide are charged dyes able
to enter damaged membranes and then excluded from viable cells, thus presenting
selectivity allowing them to be functional probes (Love et al. 2012). Trypan blue
dye is suitable for conventional spectrophotometric techniques with an absorption
peak at 605 nm, and it can generally serve for manual counting of alive/dead cells
(Monteiro-Riviere et al. 2009). On the other hand, propidium iodide is a fluorescent
dye that enters cells with disrupted membrane and intercalates into DNA and
double- stranded RNA. Suitable for a wide array of techniques, such as fluorescence
microscopy, confocal laser scanning microscopy, flow cytometry, or fluorimetry,
upon binding to nucleic acid molecules, propidium iodide fluorescence increases
20- to 30-fold (Chueh et al. 2014; Jorgensen et al. 2017). Neutral red is an uncharged
dye under physiological conditions and is retained into lysosomes of viable cells
M. C. Teixeira et al.
8.4.1 Cytotoxicity and Cell Viability Assays
Cytotoxicity and cell viability assays are the most common studies to assess the
toxicological profile for a given compound or xenobiotic. These are important in the
definition of dose–response relationship and identification of sub-lethal concentrations for mechanistic studies. Definition of dose–response curves is fundamental for
extrapolating relevant toxicological parameters, such as minimum effective concentration (MEC), half-maximal effective concentration (EC50), and maximum effect
(E max ). These parameters can be used to compare toxicological profiles of different
formulations, as well as to investigate the contribution of constitutive components
in modulating nano-based formulation toxicity (Gunsolus and Haynes 2016).
Cytotoxicity assays measure cell death after a treatment with a cytotoxic drug or
compound, and they are generally related with cell death mechanisms such as
necrosis and apoptosis (i.e., programmed and accidental cell death). These mechanisms reflect the ability of a drug molecule to trigger intracellular suicide mechanisms or destroy cells. Not very different than any other compounds or materials,
NM-induced cell death assessment focuses on examining the mitochondrial membrane integrity or potential by the assessment of the apoptotic protein levels or DNA
fragmentation (Akhtar et al. 2014). While apoptotic proteins activation are apoptosis indicators, cell membrane damage is usually considered a necrosis marker (Love
et al. 2012).
Membrane damage can be determined by measuring either the release of cytosolic molecules as lactate dehydrogenase (LDH) enzyme or the entrance or uptake
of exterior molecules that can function as probes, such as trypan blue, propidium
iodide, or neutral red dye. The LDH assay measures the activity of the enzyme
released in cell culture medium after contact with the tested NM (Jing et al. 2015).
The released LDH converts pyruvate into lactate with the consequent reduction of
NADH into NAD+. Therefore, a decrease of NADH absorption peak in extracellular medium corresponds to an increase of extracellular LDH concentration
(Caballero-Diaz and Cases 2016). Extracellular LDH content can usually be quantified by colorimetric methods based on the conversion of tetrazolium salt into soluble dyed formazan, which are also available in commercial kits (Love et al. 2012).
As previously mentioned, trypan blue and propidium iodide are charged dyes able
to enter damaged membranes and then excluded from viable cells, thus presenting
selectivity allowing them to be functional probes (Love et al. 2012). Trypan blue
dye is suitable for conventional spectrophotometric techniques with an absorption
peak at 605 nm, and it can generally serve for manual counting of alive/dead cells
(Monteiro-Riviere et al. 2009). On the other hand, propidium iodide is a fluorescent
dye that enters cells with disrupted membrane and intercalates into DNA and
double- stranded RNA. Suitable for a wide array of techniques, such as fluorescence
microscopy, confocal laser scanning microscopy, flow cytometry, or fluorimetry,
upon binding to nucleic acid molecules, propidium iodide fluorescence increases
20- to 30-fold (Chueh et al. 2014; Jorgensen et al. 2017). Neutral red is an uncharged
dye under physiological conditions and is retained into lysosomes of viable cells
M. C. Teixeira et al.