Biomedical Engineering Reference
In-Depth Information
dehydrogenase (LDH) content in extracellular mediums (Papageorgiou et al. 2007, Tkachenko
et al. 2004). Another common assay looks for the exclusion of red fluorescent ethidium homodi-
mer 1 from live cells, while measuring the uptake of calcein-AM (which fluoresces green after
modification by intracellular esterases). Assessing the level of DNA fragmentation with TUNEL
(terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling) can be used
to identify apoptosis, as demonstrated by studies on SWCNTs (Mu et al. 2009) and Eu(OH)
3
NPs
(Patra et al. 2008). It is important to consider the NM's impedance during the selection of any of
the aforementioned methods. For example, in the MTT assay, CNTs can modify the solubility of
formazan through the adsorption of the reduced crystals, thereby falsely lowering the viability
results (Worle-Knirsch et al. 2006). Such spontaneous reductions may also occur in graphene
particles (Liao et al. 2011). The LDH assay has also failed for some NPs, including Cu (LDH was
inactivated) and TiO
2
(LDH was adsorbed) (Han et al. 2011). Further advancement in this field
requires the detection and quantification of sensitive toxicological markers that may be unique to
nanotoxicity.
1.7.3
I
n
V
IVo
a
ssessMeNt
The prediction of the safety and toxicity of nanoconstructs has been examined by the extensive
testing of
in vitro
cultured cells along with
in silico
computational models.
In vivo
systems are
much more complex with interdependent pathways, which are difficult to evaluate by
in vitro
analysis. However, toxicity assays in animal models can provide better correlations with human
conditions.
Acute toxicity studies are performed in animal models to identify the maximum tolerated dose
(MTD) and no observable effect level (NOEL) in NP dosages. In classical toxicology studies, the
dose of NMs is measured by the milligrams of test items per kilogram of animal weight. However,
the surface area, size, density, and surface properties of NMs are less common to take under consid-
eration in toxicity studies. The true evaluation of nanotoxicity should be based on both the classical
mg/kg exposure and the dosage based on surface area to justify the effects of nanoscale reactivities
on toxicity. Acute toxicity studies normally span 14 days after a single dose or repeated dose admin-
istration, and the evaluation of organ-specific toxicity in addition to finding the right dose. At least
two species, one rodent and one nonrodent species, are preferably required to conclude the results
from these studies. The following parameters are monitored during the study:
•
Responses to the administered dose: Following the administration of NMs, neuronal,
hematological, and cardiac responses can occur and, hence, animals should be monitored
for at least 30 min postadministration.
•
Changes in weight: The overall health of the animal is the simplest parameter to observe
for any possible toxic effects of the injected dose. The change in weight (>10%) can sig-
nificantly indicate the NM's adverse effect. However, this is a preliminary observation and
further investigation is required to find out the actual cause of toxicity.
•
Clinical observation: The functionality of various organs systems, such as the cardiovas-
cular, respiratory, ocular, and gastrointestinal systems, are examined to evaluate clinical
changes. Imaging procedures such as ultrasound, x-ray, computed tomography (CT), and
MRI are used as supportive elements.
•
Clinical pathology: The plasma samples collected from the processed animals are utilized
to check liver functionality by the measurement of aspartate aminotransferase (AST), ala-
nine aminotransferase (ALT), and total bilirubin and albumin levels. Kidney functions are
evaluated by assessing blood urea nitrogen and creatine levels in plasma. Cardiac function
is assessed by measuring LDH and creatine phosphokinase (CPK). Amylase levels are
indicators of exocrine functions.
