This document discusses methods for evaluating the cytotoxicity of nanoparticles. It describes several common cytotoxicity assays including MTT, WST, trypan blue exclusion, and assays using dehydrogenases. The MTT assay measures mitochondrial activity and is widely used. WST assays use water-soluble reagents and do not require crystal solubilization. Dehydrogenase assays offer high sensitivity by measuring multiple cell elements. The document also provides examples of studies that used these assays to evaluate the cytotoxicity of silver nanoparticles, magnetic nanoparticles, and other nanomaterials.

1. Topic: Cytotoxicity study of Nanoparticles
Prepared and Presented By:
Aditi Chandra and Shashwat

2. • Introduction
• Methods for Cell Viability and Cytotoxicity
• Illustrations
• Reference

3.  Cytotoxicity:
It is defined as the toxicity caused due to the action of
chemotherapeutic agents to the living cells.
The Cytotoxicity tests are very important in
nanoparticles as they helps in determination of its
proposed biomedical use.

4.  Dyes
 Tritium- labeled thymidine uptake.
 MTT assay.
 WST assay.
 Dehydrogenase based assay.

6.  The method for determination of cytotoxicity and cell viability
involves dyes like trypan blue, almar blue, neutral red, coomassie
blue etc.
 The method causes differentiation of various cell in terms of colors.
The cell is differentiated on basis of cells which uptake color if they
are living or dead.
 The drawback of this method is it does not distinguishes between
cells which are dead and which are in the process of dying.

7.  Tritium- labeled thymidine uptake: Tritium-thymidine is
involved in the cell nucleus due to the cell growth, and the amount
of the tritium in the nucleus is then measured using a scintillation
counter. Though the Tritium labeled thymidine uptake assay is
sensitive to determine the influence on the DNA polymerization
activity, it requires radioisotope which causes various concerns.

8.  MTT Method: MTT is a colorimetric assay and this method is far
superior to the previously mentioned methods as it is easy-to-use,
safe, has a high reproducibility, and is widely used in both cell
viability and cytotoxicity tests. The MTT assay is the best known
method for determining mitochondrial dehydrogenase activities in
the living cells. In the method, MTT is reduced to a purple formazan
by NADH. MTT forms purple needle shaped crystals in the cells
therefore prior to measuring the absorbance, an organic solvent is
required to solubilize the crystals.

10.  WST Assay: Dojindo developed highly water-soluble tetrazolium salts
called WSTs. WSTs produce water-soluble formazans and are suitable for cell
proliferation and cytotoxicity assays. WSTs receive two electrons from viable cells
to generate a yellow, orange, or purple formazan dye. WST-8, a highly stable WST,
is often used in kits. WST-8 formazan, and 1-Methoxy PMS have no cytotoxicity in
the cell culture media, additional experiments may be carried out using the same
cells from the previous assay.

12.  Dehydrogenase Based Assay: Dehydrogenase-based assays reflect cell
conditions with more sensitivity than the other assays because they depend
on several elements including dehydrogenase, NAD(H), NADP(H), and
mitochondrial activity. The major difference between CCK-8 and the MTT
assay, other than MTT’s toxicity, is the enzymes involved. The CCK-8
assay involves most of the dehydrogenase in a cell. On the other hand,
MTT only involves mitochondrial dehydrogenase. Therefore, the MTT
assay depends on mitochondrial activity, not the cell itself. Additionally,
CCK-8 is far more sensitive than the MTT assay. Since WST-8 formazan is
water soluble, it does not form crystals like MTT. Therefore, after 1-4
hours of incubation with the CCK-8 solution, measurement of O.D. at 450
nm gives the number of viable cells. No extra steps are required.

13.  Comparative in vitro cytotoxicity study of silver nanoparticle
on two mammalian cell lines:
Silver is extensively used for its antimicrobial properties. The exact mechanism which
determines its antimicrobial property is unknown but are believed to act by reacting
with thiol group thereby making them unsuitable for microbial growth. It is also
believed to directly act by reacting with cell membrane causing membrane lysis and
thereby cell death.
The assay used for evaluation of cytotoxicity property was MTT assay method. The
absorbance of the cell was measured at 595nm and the process of assay remained the
same.
Colorimetric assay techniques like alamar blue, neutral red and coomassie blue were
also used for conduction of cytotoxic studies.

14.  Comparative In Vitro Cytotoxicity Study on Uncoated
Magnetic Nanoparticles: Effects on Cell Viability,Cell
Morphology, and Cellular Uptake
The cytotoxic studies of Magnetic Iron Oxide Nanoparticles(MIONPs) is important in
order to determine its potential biomedical applications. The MIONPs must be
biocompatible. The potential use of magnetic nanoparticles can be in areas of drug
delivery, cell-labeling, magnetofection, diagnostic imagining (MRIs) etc.
The characterization of uncoated MINOPs was done by X-ray diffraction and
Transmission Electron Microscopy ( TEM). The method for cell viability assay for
cytotoxic studies was done using staining techniques using agents like ethidium
homodimer-1.

15.  Li, L. Comparative In Vitro Cytotoxicity Study on Uncoated Magnetic
Nanoparticles: Effects on Cell Viability, Cell Morphology, and Cellular Uptake.
Journal of Nano science and Nanotechnology, 12, 1-8.
 Dechsakulthorn, F., & Hayes, A. In vitro cytotoxicity assessment of selected
nanoparticles using human skin fibroblasts. Alternatives to Animal Testing and
Experimentation, 397-400.
 Qian, Y. (2014). Enhanced cytotoxic activity of cetuximab in EGFR-positive lung
cancer by conjugating with gold nanoparticles. Scientific Reports.
 S Ebada, S. (2008). Methods for isolation, purification and structural elucidation of
bioactive secondary metabolites from marine invertebrates. Nature Protocols.

16. THE END
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