Biomedical Engineering Reference
In-Depth Information
levels of blood serum [13]. Moreover, different routes of exposure such as dermal penetra-
tion, eye absorption, or inhalation should be inspected during safety tests of nanomaterials.
Nanomaterials may enter the body by passing through skin via intercellular spaces, trans-
cellular, or trans-appendages [14]. Nanosized particles may be inhaled more deeply into the
lungs than ambient air pollution particles with approved aggressive health implications [15].
Although many different protocols have been adopted for testing nanostructures
in vitro
on
cell cultures, regulatory agencies recommend animal studies and human clinical trials to be
conducted under controlled conditions to assess the health impact of these products.
Reactions between nanostructures and biological components such as proteins or migration
of these structures through the body increases their chances for activating an immune
response [16].
Cytotoxicity Assessment
Cell death, the first step for evaluating cytotoxicity, occurs by two different processes:
necrosis and apoptosis. External factors such as infections, toxins, mechanical trauma or
physical invasions (high temperature or low oxygen pressure) result in necrosis and cytolysis,
with irreversible changes within the nuclei, cytoplasm, or organelles. Apoptosis or
programmed cell death results from induction of intercellular processes that lead to
characteristic cell changes, which include cytoplasmic shrinkage, membrane blebbing,
nuclear fragmentation, and formation of apoptotic bodies. Cell lysis triggers rapid changes
in a cell's structure, morphology, and biochemistry because of different signaling cascades.
Following membrane decomposition, cell contents are released into the extracellular
medium and can harm other neighboring cells. Early observation of cell morphology by
optical and phase-contrast microscopy is the first step for investigation of cell death in the
laboratory. However, due to limitations in optical resolution scanning electron microscopy
(SEM), transmission electron microscopy (TEM), or atomic-force microscopes (AFM) are
required for better discrimination. A TEM sample preparation protocol for visualization of
cells exposed to nanoparticles has been described by Schrand
et al
. [17]. Confocal laser
microscopy is another useful device that tracks fluorescent nanoparticles which have been
ingested by cells. Clinical conventional imaging techniques such as magnetic resonance
imaging (MRI), positron emission tomography, computed tomography, or ultrasonography
can be used to trace the
in vivo
biological performance of nanomaterials [18, 19].
A variety of techniques have been developed for
in vitro
evaluations of cell viability that can
be performed directly on cells or indirectly on cell products. These assessments can be classified
according to cell-membrane integrity, function of the organelles, or changes in DNA. It should
be noted that the type and degree of cytotoxicity responses are dependent on the targeted cell,
in addition to the particle's characteristics [20]. In the laboratory, cells can be counted by means
of a Neubauer lam (hemocytometer), where a vital dye is usually added to this procedure to
enable better discrimination between viable and nonviable cells. Trypan blue crosses the mem-
brane of dead cells, giving them a blue appearance. Leakage of the cell membrane causes release
of the enzyme lactate dehydrogenase (LDH), which results in decreased absorbance at 340 nm
due to the reduction of nicotinamide adenine dinucleotide hydrogen (NADH) to NAD
+
in the
presence of LDH [21]. Assays for viable and nonviable cells utilize fluorescent dyes to visualize
the toxicity of materials. Live cells absorb fluorescein diacetate, acridine orange, or calcein, and
fluoresce green. On the other hand, dead cells with damaged cell walls uptake propidium iodide
(PI) and fluoresce red. Other important fluorescence probes are 4ʹ,6-diamidino-2-phenylindole
(DAPI), Hoechst 33258, and Hoechst 33342, which can pass through an intact cell membrane,
bind to DNA, and cause the nucleus to fluorescence a blue color.
