Cytotoxicity analysis of vertically aligned multi-walled carbon nanotubes by colorimetric assays
Introduction
Several studies highlighted the interference of carbon nanotubes (CNT) in the optical measurements for colorimetric cytotoxicity assays. Basically there are two kinds cytotoxicity tests: with CNTs dispersed in the cell culture [1], [2], [3], [4] and; with CNTs held in some structure (films or scaffolds) in contact with the cell culture [5], [6], [7], [8].
For cells in vitro culture with dispersed CNTs, many studies suggest a low biocompatibility, while others give the opposite conclusion. Besides, remains the question if the cytotoxicity is due to CNT itself, or due to its contaminants.
The present study focuses on a new methodology to evaluate the cell viability of VACNT scaffolds produced by microwave plasma chemical vapor deposition (MWCVD) grown on Ti surfaces, without any functionalization or purification. Three different colorimetric assays were used: (i) lactate dehydrogenase (LDH), (ii) 2-(4,5-dimethyl-2-thiazolyl)-3,5-diphenyl-2H-tetrazolium bromide (MTT), and (iii) neutral red (NR). A high level of cell viability was demonstrated independent on colorimetric assays after 24 h of incubation.
Section snippets
Materials and methods
The VACNTs scaffolds were produced in thin film using a MWCVD (2.45 GHz). The substrates were Ti square pieces of 10 mm side, covered by a thin Ni or Fe layer (7 nm), both deposited by e-beam evaporator [6], [9].
L-929 mouse fibroblasts cells were provided by Cell Line Bank at Rio de Janeiro (CR019). The cells were maintained as sub-confluent monolayer's in minimum essential medium with 1.5 mM l-glutamine adjusted to contain 2.2 g/l sodium bicarbonate 85%; fetal bovine serum 15% (Gibco, BRL), 100
Results
A high density of the VACNT films grown on Fe and Ni catalysts was obtained, as shown elsewhere [6], [8]. Both cases presented a length of 6–8 μm. VACNT scaffolds produced on Fe nanoclusters are denser than those produced on Ni. No contaminants from either metallic particles or amorphous carbon were observed outside the tubes. Virtually all metallic particles are enclosed by the VACNTs scaffolds produced. The high atomic hydrogen concentration in the gas mixture efficiently removes residues of
Discussion
Multiple viability tests for several cell lines and CNT concentrations have been described in recent literature, because interferences and disturbances in colorimetric assays are likely to happen [3]. Another important point regarding toxicity is the variation of impurities of metals and amorphous carbon, from the production processes [13].
Most in vitro assays have used dispersed CNT, which is the main source of error. Woörle-Knirsch et al. used three assays to evaluate the cytotoxicity of
Conclusion
The present methodology showed that colorimetric assays are not influenced by CNT agglomerates or cell culture medium, when supported by a biocompatible substrate. Non-cytotoxic VACNT scaffolds were successfully obtained by MWCVD, without any purification or functionalization.
Acknowledgement
The authors are grateful to Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP: 2008/11642-5) for financial support.
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