213
following protonation by acidic environment. The neutral red uptake (NRU) assay
is based on the ability of viable cells to incorporate and bind neutral red dye. Since
ATP is essential for maintaining pH gradient into lysosomes, the reduced ability to
retain neutral red dye indicates cytotoxicity (Hu et al. 2015).
Cellular viability is mostly dependent on cellular damage after cell treatment
with NMs (Souto et al. 2020b, c; Silva et al. 2019a). In turn, cellular damage can
also be correlated to a reduction in the cellular metabolic activity and proliferation
(Silva et al. 2019b, c). Depending on investigated cellular process, several in vitro
assays used to evaluate cellular viability after a xenobiotic exposure (Yang et al.
2016). In vitro assays such as MTT, MTS, XTT, or WST-1, based on the cellular
reduction of tetrazolium salts to dyed formazan-based products by mitochondrial
dehydrogenases, can give a direct indication on cell viability and indirectly on cell
proliferation (Gunsolus and Haynes 2016). Differences between assays rely on
chemical composition of tetrazolium salts. While MTT is a positively charged molecule that readily enters viable cells and is converted to an insoluble formazan product, MTS/XTT/WST-1 are negatively charged molecules, which are promptly
converted into soluble formazan products. Thus, in MTT assay, formazan precipitates require previous solubilization before absorbance reading, preventing the possibility to combine different assays on the same well (Riss et al. 2011). On the other
hand, soluble formazan products obtained using MTS/XTT/WST-1 assays allow the
sequential analysis of treated cells with other in vitro assays (Riss et al. 2011).
Similar to tetrazolium salt-based cell viability assays, AlamarBlue
®
is also a proven
cell viability indicator based on the reduction of resaruzin into the bright red fluorescent resuforin by viable and metabolically active cells (Love et al. 2012).
ATP content, related to the metabolic activity of viable cells, can also be used as
a valid marker to estimate proliferation and cytotoxicity of cultured mammalian
cells (Fisichella et al. 2012; Costa et al. 2016). Luminescence-based ATP assay is a
fast, sensitive and suitable in vitro test for high-throughput screening, as the luminescent signal usually reaches a steady state within 10 min after the addition of the
specific reagent. This assay requires cell lysis, hampering further testing on the
same cells (Riss et al. 2011).
To validate in vitro assay results, proper controls should be considered. Controls
can be classified as negative (e.g., vehicle/dispersant, excipient, or soluble components) or positive (Stone et al. 2009). Negative controls are usually represented by
cell culture medium or incubation buffers such as Hank’s Balanced Salt Solution
(HBSS) (Monteiro-Riviere et al. 2009; Costa et al. 2016), and the most common
positive controls for evaluating cell viability are acetaminophen and sodium deoxycholate (Gunsolus and Haynes 2016).
8 In Vitro Methodologies for Toxicological Assessment of Drug Delivery Nanocarriers
following protonation by acidic environment. The neutral red uptake (NRU) assay
is based on the ability of viable cells to incorporate and bind neutral red dye. Since
ATP is essential for maintaining pH gradient into lysosomes, the reduced ability to
retain neutral red dye indicates cytotoxicity (Hu et al. 2015).
Cellular viability is mostly dependent on cellular damage after cell treatment
with NMs (Souto et al. 2020b, c; Silva et al. 2019a). In turn, cellular damage can
also be correlated to a reduction in the cellular metabolic activity and proliferation
(Silva et al. 2019b, c). Depending on investigated cellular process, several in vitro
assays used to evaluate cellular viability after a xenobiotic exposure (Yang et al.
2016). In vitro assays such as MTT, MTS, XTT, or WST-1, based on the cellular
reduction of tetrazolium salts to dyed formazan-based products by mitochondrial
dehydrogenases, can give a direct indication on cell viability and indirectly on cell
proliferation (Gunsolus and Haynes 2016). Differences between assays rely on
chemical composition of tetrazolium salts. While MTT is a positively charged molecule that readily enters viable cells and is converted to an insoluble formazan product, MTS/XTT/WST-1 are negatively charged molecules, which are promptly
converted into soluble formazan products. Thus, in MTT assay, formazan precipitates require previous solubilization before absorbance reading, preventing the possibility to combine different assays on the same well (Riss et al. 2011). On the other
hand, soluble formazan products obtained using MTS/XTT/WST-1 assays allow the
sequential analysis of treated cells with other in vitro assays (Riss et al. 2011).
Similar to tetrazolium salt-based cell viability assays, AlamarBlue
®
is also a proven
cell viability indicator based on the reduction of resaruzin into the bright red fluorescent resuforin by viable and metabolically active cells (Love et al. 2012).
ATP content, related to the metabolic activity of viable cells, can also be used as
a valid marker to estimate proliferation and cytotoxicity of cultured mammalian
cells (Fisichella et al. 2012; Costa et al. 2016). Luminescence-based ATP assay is a
fast, sensitive and suitable in vitro test for high-throughput screening, as the luminescent signal usually reaches a steady state within 10 min after the addition of the
specific reagent. This assay requires cell lysis, hampering further testing on the
same cells (Riss et al. 2011).
To validate in vitro assay results, proper controls should be considered. Controls
can be classified as negative (e.g., vehicle/dispersant, excipient, or soluble components) or positive (Stone et al. 2009). Negative controls are usually represented by
cell culture medium or incubation buffers such as Hank’s Balanced Salt Solution
(HBSS) (Monteiro-Riviere et al. 2009; Costa et al. 2016), and the most common
positive controls for evaluating cell viability are acetaminophen and sodium deoxycholate (Gunsolus and Haynes 2016).
8 In Vitro Methodologies for Toxicological Assessment of Drug Delivery Nanocarriers