The reliability and limits of the MTT reduction assay
for carbon nanotubes–cell interaction
Larisa Belyanskaya, Pius Manser, Philipp Spohn, Arie Bruinink, Peter Wick
*
Empa, Swiss Laboratory of Material Testing and Research, Laboratory for Materials–Biology Interactions,
Lerchenfeldstrasse 5, 9014 St. Gallen, Switzerland
Received 19 June 2007; accepted 3 August 2007
Available online 15 August 2007
Abstract
The unique physical and chemical properties of carbon nanotubes (CNTs) suggest enormous potential for many areas of research and
application. The urgent need for toxicological studies on CNTs has arisen from the steadily increasing production volume. 2-(4,5-
dimethyl-2-thiazolyl)-3,5-diphenyl-2H-tetrazolium bromide (MTT) assay is among the most versatile and popular assays used for
in vitro toxicology. For particulate samples like CNTs the applicability and limits of various tests including the MTT assay have to
be defined. Two MTT assays were applied to study the effect of single-walled CNTs (SWCNTs) on A549 cells. Both assays revealed sig-
nificant and comparable loss of cell functionality that was confirmed by DNA assay. However, using a cell-free system, we have found
that polyoxyethylene sorbitan monooleate-suspended SWCNTs interfere less with MTT assay than sodium dodecyl sulfate-suspended
SWCNTs. Moreover, depending on the purification procedure of SWCNTs, they were able to convert MTT into its MTT-formazan
insoluble form in the absence of any living system. In summary, a careful validation of MTT assay procedures is therefore suggested
in experiments where CNTs are one of the constituents, to avoid a potential bias in concluding results of cytotoxicity studies.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
Carbon nanotubes (CNTs) are a new class of materials
based on fullerene-related graphene cylinder. CNTs can
be present as single-walled (SWCNTs), double walled or
multi-walled tubes of various lengths depending on the
production procedure. Today, CNTs are produced in tons
and production volume is steadily increasing without
knowing their impact on biological systems including
humans. Their unique physical and chemical properties
suggest enormous potential for many areas of research
and application. High amounts of CNTs are used in poly-
mer nanocomposites material to increase the stiffness or
conductivity [1] as one example for CNTs future applica-
tion. Especially if CNTs are used for biomedical applica-
tions, a careful assessment of the toxicity of CNTs is
needed. Most reports on CNTs toxicity concern their
in vitro effects [2]. Several in vivo studies using mice [3,4]
and rats [5] indicated that CNTs may have a toxic potential
to human health.
Tetrazolium salts are widely used for measuring the met-
abolic activity of cells, because of the simplicity of the test
set-up. These salts are known to be reduced by various
dehydrogenase enzymes [6]. The specificity of their reactiv-
ity is defined by the positively charged quaternary tertrazole
ring core containing four nitrogen atoms. In the case of the
positively charged 2-(4,5-dimethyl-2-thiazolyl)-3,5-diphe-
nyl-2H-tetrazolium bromide (MTT) one of the aromatic
groups is replaced by a thiazolyl ring [7]. The net positive
charge of MTT supports the cellular uptake via the plasma
membrane. MTT is thought to be nearly exclusively
reduced into its formazan intracellularly by a variety of
cytoplasmic substrates including NAD(P)H-oxidoreduc-
tases but also by the mitochondrial enzyme succinate dehy-
drogenase. The amount of formazan formed is directly
proportional to the number of metabolically active cells in
the culture, which can be quantified spectrophotometrically
0008-6223/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carbon.2007.08.010
*
Corresponding author. Fax: +41 71 274 76 94.
E-mail address: peter.wick@empa.ch (P. Wick).
www.elsevier.com/locate/carbon
Available online at www.sciencedirect.com
Carbon 45 (2007) 2643–2648