Biomedical Engineering Reference
In-Depth Information
have different biodistribution profiles, biological accumulation and interactions, and physical and
chemical clearance processes. Understanding the relationship between the physicochemical prop-
erties and their ADME characteristics is critical in achieving desired biological effects and also
minimizing potential toxicities. On the other hand, a number of nanoparticles promote the forma-
tion of pro-oxidants, and, subsequently, impair the balance between the biological system's ability
to produce and detoxify reactive oxygen species (ROS). The increased levels of ROS can result in
cell apoptosis or necrosis by reacting with DNA, proteins, carbohydrates, and lipids in a destruc-
tive manner. Primary genotoxicity can be a result of the direct exposure to nanoparticles, while
secondary genotoxicity can be related to the interactions of nanoparticles with cells or tissues, and
the adverse effects, like inflammation and oxidative stress, caused by factors released via these
interactions.
Considering the specific biological characteristics of nanoparticles, multidisciplinary studies are
encouraged in establishing nanoparticle classifications and testing procedures emphasizing safety
regulations.
10.3
IN VITRO
EVALUATION OF NANOPARTICLE TOXICITY
In vitro
studies are conducted using established cell lines and primary cells derived from target tis-
sues. As there is an absence of clear guidelines about how to test and evaluate nanoparticles,
in vitro
studies are extremely important to provide information on the toxicity of nanoparticles. Generally,
for nanotoxicological studies, the experimental techniques are the same as the ones employed for
biological studies and toxicological studies. There are seven main categories of traditional
in vitro
studies, including both genomic methods and conventional methods.
10.3.1
I
n
V
Itro
c
ytotoxIcIty
s
creeNINg
A variety of approaches could be applied to determine the damage from nanoparticles on cell via-
bility. It is important to choose an appropriate cytotoxicity assay when testing, since nanoparticles
can influence results by adsorbing dyes or play roles in redox reactions. For example, in terms of
the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, the over- or under-
estimation of cell viability may happen due to the characteristics of nanoparticles. Overestimation
is probably caused by the particles, which generate an absorbance at the same wavelength as that
used to quantify the colored product, and underestimation may be because of the large surface area,
or other surface properties, causing a high adsorptive ability that allows the nanoparticles to effec-
tively extract the colored product from the cell extract (Worle-Knirsch et al., 2006). Considering the
above possibilities, a positive- and negative-control particle should be included as a benchmark to
the tested nanoparticle. Moreover, a suspension containing cell debris, the dissolved formazan, and
particulates would be generated when testing nanoparticles. Thus, in order to read the absorbance of
the supernatant without particles, cell debris, and other background interference, the centrifugation
and transfer of the sample to a fresh, 96-well plate is recommended. Table 10.1 summarizes several
of the most commonly used cytotoxicity assays.
10.3.2 a
ssays
of
ros
ROS are electrophilic molecules or free radicals containing an oxygen atom. They can occur natu-
rally in the body as intermediates in metabolic reactions, or as a result of toxic insults.
One of the most common ways to assess the roles of nanoparticles in oxidative stress is to use
the fluorescent marker H2DCF-DA (2′, 7′-dichlorodihydrofluorescein diacetate). Fluorescence
increases when H2DCF-DA is oxidized to DCF by ROS (2′, 7′-dichlorofluorescein). This assay is
carried out to test the toxicity of various nanoparticles such as Ag-NPs (Singh and Ramarao, 2012),
TiO
2
, Fe
2
O
3
, Co
3
O
4
, Mn
3
O
4
(Limbach et al., 2007), and so on.
