Biomedical Engineering Reference
In-Depth Information
19.9.2.4 Nitrite Production
Nitrite production is determined by the Griess reagent (1:1 mixture of 1% sulfanilamide in 5% phos-
phoric acid and 0.1%
N
-1-naphthylethylenediamine dihydrochloride). In this method, the Griess
reagent is mixed with culture supernatant and the intensity of color is determined at a particular
wavelength using ELISA plate reader [135].
19.9.2.5 Mitochondrial Membrane Potential Dissipation Assay
The changes of mitochondrial membrane potential (MMP) after exposure of NPs are determined
by the uptake of tetramethyl rhodamine ethyl ester (TMRE). TMRE is a red-orange fluorescent
permeable cationic, lipophilic dye. It can be readily taken up by active mitochondria into negatively
charged mitochondrial matrix. The intensity of fluorescent obtained is indicative of the MMP [165].
19.9.3 I
NflaMMatory
r
espoNse
The
in vitro
method to evaluate the inflammatory response has been performed via ELISA. The
ELISA method was first described in 1971, and enables simple and accurate quantification of inflam-
matory markers in cell culture supernatants through antibodies and enzymatic detection reactions
[166]. ELISA results have been reported for NPs of different composition and origin, for example,
for titanium dioxide [167], iron oxide [168], zinc oxide [169], carbon black [170], CNTs [171], fuller-
enes [172], silica [173], and for QDs [174]. The most commonly tested human and murine inflam-
matory markers are the chemokine interleukin-8 (IL-8), followed by TNF-α and IL-6. In some
cases, IL-1β as well as a few other cytokine and stimulating factor concentrations are measured.
The chemokine MIP-2 is usually quantified in rat model systems together with TNF-α and/or IL-6.
Recently, NPs have been reported to interfere with enzymatic immunoassays. Adsorption of
cytokines by NPs
in vitro
was discussed for carbon NPs (IL-8, [175]) and for metal oxides (IL-6)
[176]. Even depletion of trace nutrients or growth factors from cell culture media due to the high
adhesive surface area of NPs was considered [175,177].
19.9.4 g
eNotoxIcIty
Nanogenotoxicology is yet another new term that was coined to represent the growing trend of
research into NP-induced genotoxicity and carcinogenesis. Although there are still no conclusive
links with NP-induced genotoxicity and lung cancer from past epidemiological studies and
in vivo
rodent experiments, some researchers have pointed out that long-term inflammation and oxidative
stress present in tissue environment eventually induces DNA damage in cells and tissues [178]. This
is of particular concern, especially if the NPs continue to generate an oxidative environment in the
cell that causes gene mutations/deletions. This can lead to larger-scale mutagenesis and carcino-
genicity, and subsequently, the development of tumors and cancer [156]. Already, more evidence
has emerged regarding the DNA-damaging properties of certain classes of NPs, particularly, the
metal-based NPs such as Ag NPs, Au NPs, and TiO
2
NPs [179-181]. One proposed mode of action
for NP genotoxicity is the ability of signaling peptides functionalized on NPs such as CNTs that
enable them to enter the nucleus via nuclear pores [182]. It is yet to be shown that such CNTs are
able to cause genotoxicity, but it is believed that there is a greater potential of damage when NPs
are able to get in close proximity to DNA. There are also other different mechanisms that may be
specific to the elemental composition and shape of NPs, which could lead to DNA damage such
as single-strand breaks, double-strand breaks, DNA deletions, and genomic instability in the form
of increase in 8-hydroxy-20-deoxyguanosine levels [86]. While some researchers have found that
exposure to TiO
2
NPs in rats could cause formation of lung granulomas [159], others have cautioned
that appearance of granulomas does not necessarily mean that the tissue is cancerous as most tissues
probably remain benign [183]. As most reports regarding NP toxicity were observed from experi-
ments involving UV or irradiation exposure, the clinical relevance of these mechanistic experiments
