Biomedical Engineering Reference
In-Depth Information
and PEG-silane modified CdSe/ZnS QDs (Zhang et al. 2006) on the proliferation of HEK293 and
human lung and skin epithelial cells has been assessed by FCM. The CFE assay has been used to
assess the effects of polymeric-entrapped, thiol-coated Au nanorods (Zhang et al. 2006) on murine
fibroblasts and human hematopoietic progenitor cells.
1.7.2.5 Exocytosis
Changes in exocytosis may be another indicator of nanotoxicity. Carbon-fiber microelectrode
amperometry has been employed to study the effects of various NPs on the secretion of small,
electro-active molecules (e.g., serotonin and epinephrine). This method allows one to quantify the
number of chemical messenger molecules released per vesicle, the specific release kinetics, and the
frequency of vesicle fusion with a high sensitivity and time resolution. Studies in MPMCs and adre-
nal chromaffin cells have utilized this method to reveal the mutagenic potential of functionalized
(with either positive or negative side chains) Au and Ag NP exposure (Marquis et al. 2011).
1.7.2.6 Cell Viability and Metabolic Activity
Cell viability studies are perhaps some of the most widely used assays to assess nanotoxicity, as
they provide information on the mechanisms or causes of cellular toxicity and death. Any lethal
consequences from NP exposure, including membrane lysis, cell cycle arrest, and apoptosis, may
stop mitochondrial activity. Many different types of assays that allow for the study of toxicity are
used in research. Toxicity can also be assessed by using two or more independent test systems to
validate findings. The colony formation assay, or the clonogenic assay, is an
in vitro
cell-survival
assay, based on the ability of a single cell to grow into a colony. It is a simple method that can be
employed to avoid interference from NPs, as no dye or stain is used (Franken et al. 2006).
Assays of metabolic activity following exposure to NPs are the most common methods used to
determine cell viability. The most popular test is the MTT assay in live cells. MTT [3-(4,5-dimeth-
ylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] is reduced to purple formazan, which can be
detected spectrophotometrically. Several similar assays (MST, MTS, XTT, WST-1) have also been
employed to eliminate the possibilities of NM interference with these assays (Stone et al. 2009).
Alamar blue (resazurin) is another dye that undergoes reduction by living cells to produce the fluo-
rescent product, resorufin. It has also been extensively utilized to measure cell viability, following
exposure to SiO
2
-coated CdSe QDs (Sharma et al. 2009) and amino acid-functionalized Au (Ghosh
et al. 2008).
1.7.2.7 Hemolysis
The risk of erythrocytic lysis is especially important for NPs that are intended to be directly intro-
duced into the bloodstream. The assessment of hemoglobin (Hb) by spectrophotometric techniques
in response to NP exposures can be a measure of both membrane disruption and extreme cellular
toxicity (i.e., necrosis). This approach has been utilized to determine the median lethal dose values
for functionalized Au NPs (Goodman et al. 2004). Recent studies have focused on the hemolytic
potential of functionalized Au NPs while assessing their effects on ROS production in neutrophils
and thrombotic capabilities (Love et al. 2012).
The assessment of the indicators of programmed cell death (i.e., apoptosis) and necrosis directly
reveal a NP's ability to induce intracellular, self-destruction mechanisms and destroy cells. Such
assays have been developed beyond the measurement of membrane integrity to the quantification
of apoptotic protein levels and activation and DNA fragmentation. There are five main tech-
niques generally used to determine membrane integrity: phosphatidylserine (which migrates to
the extracellular surface of apoptotic cells) labeled with annexin V, propidium iodide exclusion by
intact membranes (AshaRani et al. 2009), trypan blue exclusion by intact membranes (Goodman
et al. 2004, Hauck et al. 2008), neutral red staining (which undergoes a color change due to
protonation in intact lysosomes) (Lanone et al. 2009), and the determination of the total lactate
