Advanced Materials Research Vols. 287-290 (2011) pp 32-36
Online available since 2011/Jul/04 at www.scientific.net
© (2011) Trans Tech Publications, Switzerland
doi:10.4028/www.scientific.net/AMR.287-290.32
Cytotoxicity of Single-walled Carbon Nanotubes With Human Ocular
Cells
Lu Yan,1,2,4 Shu Zhang,1,2,4 Chao Zeng,1,2 Yuhua Xue,1,2 Zhonglou Zhou,1
Fan Lu,1 Hao Chen,1,2 Jia Qu,1,2 Liming Dai,1,2,3,a and Yong Liu1,2,b
1
School of Ophthalmology and Optometry, Wenzhou Medical College
2
Institute of Advanced Materials for Nano-Bio Applications, Wenzhou Medical College
Wenzhou, Zhejiang 325027, China
3
Department of Chemical Engineering, Case Western Reserve University
Cleveland, Ohio 44106, USA
4
These authors contributed equally
a
ldai@mail.eye.ac.cn, byongliu1980@hotmail.com
Keywords: Single-walled Carbon Nanotubes (SWCNTs), Ocular Biocompatibility, Human RPE
Cells, CCK-8 Assay, SOD Assay, LDH assay, Apoptosis Assay
Abstract. In this paper, we report the first study on cytotoxicity of single-walled carbon nanotubes
(SWCNTs) and theirs derivatives with human ocular cells, such as ARPE-19 cells. In particular, we
have systematically investigated the cytotoxicity of SWCNTs, hydroxyl-functionalized SWCNTs
(SWCNT-OH), and carboxylic functionalized SWCNTs (SWCNT-COOH) with ARPE-19 cells by
examining their influence on the cell morphology, viability, oxidative stress, membrane integrity and
apoptosis. To this end, various methods, including optical micrography, CCK-8 assay, LDH assay,
SOD assay, TEM and Apoptosis assay, have been used in this study. Our results suggest that
SWCNTs could cause an decrease in the cell survival rate, changes in the SOD level, membrane
integrity and cell apoptosis, indicating a high toxicity to ARPE-19 cells. However, chemical
functionalization of SWCNTs with –OH and –COOH groups was found to significantly improve the
biocompatibility of SWCNTs. Among the SWCNTs and their derivatives studied in this work, the
SWCNT-COOH exhibits the best biocompatibility to ARPE-19 cells.
Introduction
Carbon nanotubes (CNTs) have attracted intensive attention during recent years [1]. Due to their
unique thermal, mechanical and electrical properties, CNTs have potential for a wide range of
practical applications in physical, chemical, mechanical, electrical, and biomedical areas [2-8]. Of
particular of interest, SWCNTs have been used as potential components of novel biomaterials [9, 10].
Along with the development of nanotube composites for commercial utilization, it is essential to
evaluate the influence of SWCNTs on environment and human health. Recently, much effort has been
made on cytotoxicity and genotoxicity of SWCNTs both in vitro and in vivo. Cytotoxicity of
SWCNTs has been determined via various exposure ways, such as skin touch, ingestion, intravenous
injection and inhalation [11-15]. Ocular toxicological effects of SWCNTs, however, have not been
reported yet. Eyes are special organ in a human’s body. Eye exposure to SWCNTs is inevitable during
ordinary delivery or practical utilization of SWCNTs. It is thus essential to evaluate the ocular toxicity
of SWCNTs either in vitro or in vivo. In this paper, we report for the first time our results on
cytotoxicity of SWCNTs with human retinal pigment epithelium (RPE) cells. RPE, a main
component in maintaining the integrity of the retinal structure and function, is essential to the survival
of photoreceptors. ARPE-19, a cell line derived from human RPE, has been used to determine ocular
biocompatibility of SWCNTs. We further measured cytotoxicity of SWCNTs functionalized with
various functional groups, including hydroxyl (SWCNT-OH) and carboxyl (SWCNT-COOH), to
enhance the biocompatibility of SWCNTs for practical applications.
All rights reserved. No part of contents of this paper may be reproduced or transmitted in any form or by any means without the written permission of TTP,
www.ttp.net. (ID: 129.22.1.19, Case Western Reserve University, Cleveland, United States of America-04/06/13,19:17:09)
Advanced Materials Research Vols. 287-290
33
Experimental
SWCNTs and SWCNT-OH were obtained from Shenzhen Nanotech Port Co. Ltd. Typically, the
diameter of SWCNTs is less than 2nm and their length is around 5-15µm. SWCNT-COOH was
prepared from the acid oxidation of SWCNTs. Dulbecco’s Modified Ealge’s Medium (DMEM), Fetal
Bovine Serum (FBS), and trypsin were all purchased from Invitrogen Corporation, while Cell
Counting Kit -8 (CCK-8) and SOD Assay Kit-WST were from Dojindo Molecular Technologies, Inc.
Lactate Dehydrogenase (LDH) Release Test Kit (CytoTox 96® Non-Radioactive Cytotoxicity Assay)
was provided by Promega Corporation. Hoechst Staining Kit was supplied by Beyotime
Biotechnology. All other chemicals were purchased from Sigma-Aldrich.
ARPE-19 cells were seeded in 96-well plates at the density of 5000 cells/well with DMEM/F12
medium containing 10% FBS. Cells were cultured in the incubator with various concentrations
(5µg/ml, 10µg/ml, 50µg/ml, 100µg/ml) of SWCNTs or their derivatives for 24, 48, 72 hours,
repectively. Cells cultured in the medium without addition of SWCNT containing materials were
used as the control. All ARPE-19 cells were cultured at 37°C in a 95% air/5% CO2 humidified
incubator.
Results and Discussion
Fig. 1 shows FTIR spectra of functionalized SWCNTs compared with the pristine SWCNTs. The
band at 3400cm-1 found in the spectrum of SWCNTs (black line, Fig. 1) can be attributed to the
vibration mode of hydroxyl groups. This band was found to be sharper and stronger for SWCNT-OH
and SWCNT-COOH, suggesting an increased content of –OH upon chemical functionalization of
SWCNTs. The band associated with the stretching vibration of carbon-oxygen bonds could be
observed around 1680 cm-1 for the SWCNT-OH and SWCNT-COOH (red and blue curves,
respectively, in Fig. 1) whereas it was negligible in the pristine SWCNTs. Although there is no
obvious difference in FTIR spectra for the SWCNT-OH and SWCNT-COOH, they do show different
cytotoxities as to be seen below.
Fig. 1. FTIR spectra of SWCNTs (black), SWCNT-OH (red) and SWCNT-COOH (blue).
A fast and efficient signal of cell status can be obtained from cell morphology. Well shaped
morphologies were observed for ARPE 19 cells without the treatment with SWCNTs (Fig 2a) and
incubated with 25µg/ml of SWCNTs for 72 hours (Fig. 2b). No obvious change in cell morphology
was found for ARPE-19 cells either incubated with 25µg/ml of SWCNT-OH (Fig 2c) or
SWCNT-COOH (Fig 2d) under the same condition for 72 hours, suggesting a minimal influence of
the SWCNT based nanomaterials on the morphology of ARPE-19 cells. Well dispersion of
SWCNT-COOH in culture supernatants can be observed in Fig. 2d.
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Applications of Engineering Materials
Fig. 2. Optical micrographs of ARPE-19 cells after 72h incubation (a) without SWCNTs (the control),
(b) with 25µg/ml SWCNTs, (c) with 25 µg/ml SWCNT-OH, and (d) with 25 µg/ml SWCNT-COOH.
Cell survival rates of ARPE-19 cells incubated with SWCNT based nanomaterials were
determined using CCK-8 assay. Fig. 3 shows that the survival rate of ARPE-19 cells decreased with
increasing concentration of the SWCNT containing nanomaterials and culturing time. We also found
that SWCNT-COOH exhibited the lowest toxicity to ARPE-19 cells in comparison with SWCNTs
and SWCNT-OH (Fig. 3). SWCNT-OH showed a higher biocompatibility than the pristine SWCNTs,
but lower than SWCNT-COOH. The observed relatively lower toxicity for functionalized SWCNTs
may be attributed to their improved dispersibility induced by the functional groups.
ARPE-19-48h
Cell Viability(%)
ARPE-19-72h
(b)
SWCNT
SWCNT-OH
SWCNT-COOH
100
90
80
70
60
50
40
30
20
10
SWCNT
SWCNT-OH
SWCNT-COOH
100
Cell Viability(%)
(a)
90
80
70
60
50
40
30
20
10
0
0
5
10
50
Concentration(μg/ml)
100
5
10
50
100
Concentration(μg/ml)
Fig. 3. Cell survival rate assay of ARPE-19 cells incubated with various amounts of SWCNTs
(black), SWCNT-OH (red) and SWCNT-COOH (blue) for (a) 48h and (b) 72h, respectively.
Superoxide dismutase (SOD), which catalyzes the dismutation of the superoxide anion (O2-) into
hydrogen peroxide and molecular oxygen, is one of the most important antioxidative enzymes. SOD
Assay is generally used for detection of oxidative stress level in cells. As shown in Fig. 4a, both the
SWCNTs and SWCNT-COOH exhibited very low toxicity at all concentrations (up to 100µg/ml)
investigated in this study while SWCNT-OH showed significant toxicity at the high concentration
(100µg/ml). These results can also be confirmed by the cell survival rate assay (Fig. 3) and LDH
results (Fig. 4b). Therefore, the oxidative stress may cause certain cell injury and the observed slight
cell viability decrease at high concentration of SWCNT-OH, as reported elsewhere [16, 17]. LDH
release level reflects the cell membrane integrity. As can be seen in Fig. 4b, LDH release caused by all
types of SWCNTs were well below25%, indicating that the membrane integrity of ARPE-19 cells was
largely maintained. The lowest LDH release level was found for SWCNT-COOH at the high
concentration (more than 50µg/mL) while the highest LDH release was obtained from the pristine
SWCNTs. The LDH result is in a good consistance with the cell survival rate assay and SOD assay. It
is also interesting to observe that the LDH release levels of the functionalized SWCNTs decreased
Advanced Materials Research Vols. 287-290
35
with increasing concentration above 10µg/mL. This probably indicates that SWCNT-OH and
SWCNT-COOH have been swallowed by cells at relatively high concentration without causing any
damage on the cell membrane, though it cannot be ruled out that leakage tunnels of cells were
partially blocked by aggregates of the functionalized SWCNTs at high concentrations [18] .
Fig. 4. (a) SOD assay of SWCNTs (black), SWCNT-OH (red), SWCNT-COOH (blue) in ARPE-19
cells and (b) LDH assay for detection of ARPE-19 cells incubated with SWCNTs (black),
SWCNT-OH(red) and SWCNT-COOH (blue), respectively, for 72h.
To visulize the nanotube-cell interaction, APRE-19 cells were incubated for 72h in the presence and
absence of 50µg/ml SWCNT-COOH prior to be harvested and washed using phosphate buffer
solution (PBS) (pH 7.4). These cells were subsequently washed again using 0.1M PBS and fixed for
4h using 2.5% glutaraldehyde prior to TEM examination. TEM images given in Fig. 5 clearly show
the presence of SWCNT-COOH in ARPE-19 cells. As shown in Fig. 5, organelles, including
mitochondria, ER and Golgi, were almost dissolved, suggesting that SWCNT-COOH penetrated into
the cytoplasm through the cell membrane by swallowing[19, 20]. However, it is also possible that
SWCNT-COOH might have damaged the cell membrane and transferred into the cell through the
damged cell membrane, albeit less likely as indicated by our LDH data. Similar results were obtained
from the SWCNTs and SWCNT-OH.
Fig. 5. TEM micrographs of ARPE-19 cells (A) treated with SWCNT-COOH for 72h, and (B) at a
higher magnification for the selected areas of A. White arrows in (B) pointing to SWCNT-COOH.
We further measured the effect of SWCNTs, SWCNT-OH and SWCNT-COOH on apoptotic cells
using fluorescence microscopy after staining the cells with Hoechst 33258 (Fig. 6). Apoptotic cells
exhibited condensed nucleus with a round shape and enhanced fluorescent signal (white arrow in Fig.
6), indicating that SWCNTs, SWCNT-OH and SWCNT-COOH all induced some apoptosis or
necrosis.
Fig 6. Fluorescence micrographs of Hoechst 33258 stained ARPE-19 cells incubated (a) without
SWCNTs, (b) with SWCNTs, (c) with SWCNT-OH and (d) with SWCNT-COOH. White arrows
indicate cell apoptosis. (Scale bar: 50µm)
36
Applications of Engineering Materials
Conclusions
We have investigated cytotoxicity of SWCNTs, SWCNT-OH and SWCNT-COOH with human RPE
cells using various techniques, including optical micrograph, CCK-8 assay, SOD assay, LDH assay,
TEM and Apoptosis assay. It was found that SWCNTs could cause a decrease in the cell survival rate
with certain adverse effects, such as apoptosis and necrosis. SWCNTs were found to enter inside cells
by uptake or cell membrane damage. Our results also suggest that eye protection is necessary when
dealing with SWCNTs. Although SWCNTs exhibited significant toxicity to ARPE-19 cells,
functionalized SWCNTs, such as SWCNT-OH and SWCNT-COOH, showed much lower toxic than
the pristine SWCNTs. Among the SWCNTs and their derivatives studied in this work, the best
biocompatibility was obtained from SWCNT-COOH, suggesting a potential method to improve
biocompatibility of SWCNTs via functionalization for practical applications.
Acknowledgements
Authors are grateful to Dr. Lin Hou (Wenzhou Medical College) for kindly providing ARPE-19 cells.
This work was financially supported by the National “Thousand Talents Program”, the Ministry of
Science and Technology of China (2009DFB30380), the Zhejiang Department of Science and
Technology (2009C13019), the Chinese National Nature Science Foundation (81000663) and the
Zhejiang Department of Education (Y200906587).
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Applications of Engineering Materials
10.4028/www.scientific.net/AMR.287-290
Cytotoxicity of Single-Walled Carbon Nanotubes with Human Ocular Cells
10.4028/www.scientific.net/AMR.287-290.32