Biomedical Engineering Reference
In-Depth Information
methods for quantification of cell viability are available, which differ in sensitivity, reliability, and
ease of use. These assays are based on three basic parameters. The first assay type is based on the
measurement of cellular metabolic activity. An early indication of cellular damage is a reduction
in metabolic activity. The MTS colorimetric assay is a commonly used assay of this category.
In this assay, living (metabolically active) cells reduce tetrazolium salts (3-(4,5-dimethylthiazol-
2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2
H
-tetrazolium, inner salt; MTS) and the
electron-coupling reagent, phenazine methosulfate to formazan, which is soluble in tissue culture
medium. The absorbance of the formazan product formed can then be determined at 492 nm.
Since the production of formazan is proportional to the number of living cells, the intensity of
the color produced is a good indicator of cell viability [94]. Another set of cytotoxicity assays is
based on the measurement of membrane integrity. The cell membrane forms a functional barrier
around the cell, and traffic into and out of the cell is highly regulated by transporters, receptors,
and secretion pathways. When cells are damaged, they become “leaky” and this forms the basis
for the second type of assay. Lactate dehydrogenase (LDH) is a stable cytoplasmic enzyme that
is present in most cells. This enzyme cannot be measured extracellularly unless cell damage has
occurred. Thus, membrane integrity can be determined by measuring LDH in the extracellular
medium. The amount of enzyme activity correlates to the proportion of damaged cells [95]. Cell
membrane integrity can also be assessed by the uptake or exclusion of dyes, such as trypan blue
or eosin. These dyes are normally excluded from the inside of healthy cells; however, if the cell
membrane has been compromised, they freely cross the membrane and stain intracellular com-
ponents. The third type of assay is the direct measure of cell number, since dead cells normally
detach from a culture plate, and are washed away in the medium. Cell number can be measured by
direct cell counting, or by the measurement of total cell protein or DNA, which are proportional
to the number of cells [96].
7.4.2.2 Cell Uptake Studies of Nanoparticles
Cell uptake study can be done by using the following techniques:
1. Flow cytometry (including FACS)
2. Epi-fluorescence microscopy
3. Confocal microscopy
To study cellular uptake, appropriate cell lines such as the human ovarian carcinoma cell line
and the human epidermal carcinoma cell line are selected and cellular accumulation is measured.
As all these methods are fluorescence-based methods, suitable dye is attached to the targeting moi-
ety and its uptake is studied [97].
7.4.2.3 Assays for Alteration in Gene Expression
Gene expression assays, that is, gene profiling, is an important tool for screening different envi-
ronmental particles, including nanoparticles. Techniques used to assess gene expression include
Northern blot analysis, ribonuclease protection assays, quantitative real-time polymerase chain
reaction, polymerase chain reaction arrays, and microarrays.
7.5 CONCLUSION
PNs and SLNs are widely used in the pharmaceutical industry as drug delivery systems. They are
usually regarded as safe and very few studies have been done to determine the nanotoxicity of
these systems. Since PNs and SLNs are parallel to different nanoparticulate systems (like inorganic
nanoparticles showing nanotoxicity) and possess similar properties like particle size, it is required
that the nanotoxicity of these systems should be studied in detail to fully exploit the advantages
offered by PNs and SLNs.
