Jpn. J. Protozool. Vol. 40, No. 2. (2007)

139

Original

Paramecium as a bioassay system for elucidation of cytotoxicity and biocompatibility of nanoparticles: effects of carbon nanofibers on proliferation and survival
Nobuyuki HAGA1* and Koichi HANEDA2
1

Department of Biological Technology, 2Department of Information Technology and Electronics, Senshu University of Ishinomaki, Ishinomaki, Miyagi 986-8580, JapanSUMMARY
Carbon nanofibers (CNF), composed of carbon
nanotubes, are a recent technological advance withwide applications in nano-engineering fields including biotechnology and biomedicine. However,
little is known about the environmental effects of
CNF, or their potential danger to human health. To
elucidate the safety of CNF, we examined the cytotoxicity of CNF in Paramecium. In this study weconsidered the cytotoxicity effect of CNF in two
categories of cellular properties, cell survival andcell proliferation. We show that CNF are ingested
and concentrated as efficiently as nutritive bacteria
by paramecia, revealing a means by which CNF
could be introduced into the food webs of aquatic
ecosystems. Clear cytotoxicity of CNF was detected in survival tests by extracellular application*Corresponding author
Tel: +81-225-22-7716, Fax: +81-225-22-7746
E-mail: haga@isenshu-u.ac.jp
Received:5 March 2006; Accepted:10 September 2007

at extremely high concentration (up to 50 mg/ml)
in culture medium containing nutritive bacteria.
Contrary to this effect, no cytotoxicity was detected in survival tests using the modified Dryl’s
solution (K-DS) that is used as a buffered saline ofculture medium. The cytotoxicity of CNF suggests
an interaction between CNF and the components inculture medium or their metabolic products produced by digestion of components in culture medium. Another cytotoxicity effect was detected inproliferative activity test at lower concentrations of
CNF (up to 500 µg/ml). The cytotoxicity on proliferative activity was reversible and recovery occurred within 24 hours after removal of CNF. Our
results suggest that Paramecium is useful for abioassay of nanoparticle cytotoxicity. For the elucidation of safety of CNF we have to examine boththe optimum concentration of CNF and co-existingbiomaterials in a test solution. In conclusion, CNF
have a high potential for cytological and biomedical application under precise control of concentration.

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Cytotoxicity test of nanoparticles using Paramecium

INTRODUCTION
The safety of materials to living organisms usually depends upon particle size: substances that aresafe in macroscopic quantities can become dangerous when reduced to microscopic particles (Brown
et al, 2001). As the behavior of nanoparticles in theenvironment and in cells is not well understood, it
is important to establish an experimental system inwhich to assess the effects of nanoparticles on theenvironment and on living organisms.
Paramecium, a ciliated eukaryotic unicellularorganism that lives in fresh water, has several features that make it a potentially valuable model toassay the cytotoxicity of environmental agents(Rajini et al, 1989, Smith-Sonneborn et al, 1983,1986): they can ingest both soluble molecules andmany different types of particle, including polyethylene particles, India ink, iron filings and sand, byphagocytosis, and they show very stable cellularfunctions such as phagocytosis, cell division andswimming behavior. In this study, we examine theuse of Paramecium as a bioassay for evaluating
the cytotoxicity of nanoparticles and have established a standard experimental method that enables
micro liter-sized assays.
The CNF used in this experiment have a structure of hollow graphitic tubules of nanometer dimensions, and are comprised of coaxial tubes ofhelically arranged carbon-hexagon sheets. Typically, their diameters ranged from 2 to 20 nm, andthey were several micrometers in length (Iijima,1991).

MATERIALS AND METHODS
Cells and culture medium
Paramecium caudatum, syngen 3 was used inthis study. They were grown at 25°C in a culture
medium containing 1.25% (w/v) fresh lettuce juice

diluted with K-DS (Dryl’s solution (Dryl, 1959)
modified by substituting KH2PO4 for NaH2PO4,
pH 7.0 (Yanagi, 1987)) and inoculated with Klebsiella pneumoniae one day before use (Hiwatashi,1968).
CNF production
CNF were provided by Prof. K. Tohji (Tohoku
University, Japan). They were produced in a conventional flow reactor system (Rodriguez, 1993) inwhich a hydrocarbon/carrier-gas mixture (C2H4/H2(4:2)) was pyrolysed at 873 K for 4 hours in thepresence of powdered Ni catalyst. The catalyst
was positioned in an Al2O3 boat and set in a quartz
tube assembled in a horizontal tube furnace; it had
been reduced in advance in a 10% H2/He stream
for 2 hours at 873 K.
As the CNF inevitably contain an appreciable
amount of metallic elements, such as Ni, a purification process is vital to both fundamental studies
and potential applications of the material (Ebbesen
et al, 1994; Tohji, 1996). The CNF used in thisstudy were purified by hydrochloric acid treatment(Tohji et al, 1996).
CNF ingestion
CNF were washed twice with K-DS with centrifugation at 10,000 rpm for 5 min. The washed
CNF were suspended at a concentration of 100
mg/ml in K-DS. Paramecia were washed twicewith K-DS by a hand centrifuge and suspended in
K-DS at the cell density of about 4,000 cells/ml.
Then, 40 µl of the CNF suspension was mixed
with an equal volume of the cell suspension andthe mixture was incubated at 25°C. The paramecia
were periodically isolated from the mixture with ahand-made glass micropipette and put into a dropof 2.5%(w/v) methyl cellulose dissolved in K-DS
on a glass slide. Photographs were taken under alight microscope (ZEISS AX10) by a digital camera (Nikon, DS-5Mc).

Jpn. J. Protozool. Vol. 40, No. 2. (2007)

Cytotoxicity test of CNF
A CNF suspension was prepared in an autoclaved glass depression slide with an autoclaved
microchip by mixing CNF stock suspension witheither K-DS or fresh culture medium. Then, a single paramecium was added into 100 µl of the CNF
mixture with a hand-made glass micropipette under a binocular microscope. The CNF suspension
with the paramecium was incubated at 25°C.
Capillary culture method
We developed a small-sized bioassay using a
glass capillary (0.6 mm inside diameter and 35 mm
length) by modification of the method (Haga and
Hiwatashi, 1981). Twenty µl of a CNF suspension
containing paramecia was put on a glass depression slide and then the suspension was drawn intoa capillary by capillary action. The capillary wasplaced in a laboratory dish with a sheet of wet paper. This method allows a one-week incubation at
25°C without any serious problem of evaporationof the medium.

141

Statistic analysis
Statistical analysis of the data from the doseeffect experiments was performed using the Dunnett test (Graph pad Prism 4).

RESULTS
Ingestion of CNF and food vacuole formation
Paramecia ingest nutritive bacteria via the buccal cavity and concentrate the bacteria, together
with a small amount of fluid, in food vacuoles.
CNF-containing food vacuoles are shown in Figure 1a and 1b. Immediately after initial contact
with CNF Paramecia began to ingest CNF by
forming a food vacuole. CNF particles were concentrated in each food vacuole. No CNF particle
that escaped from a food vacuole into cytoplasm
was detected by a light microscope observation.
Effect of CNF on non-proliferating cell survival
Before testing the cytotoxicity of CNF, we examined experimental conditions to prepare homogeneous dispersion of CNF suspension in K-DS.
By varying the concentrations of CNF and the timeof sonication, we confirmed that a homogeneous
suspension of CNF could be obtained in up to aconcentration of 50 µg/ml in K-DS for 20 min
sonication.
Paramecia do not undergo proliferation whenincubated in a bacteria-free solution. The survivalof non-proliferating paramecia was examined byusing a capillary culture with 20 µl of CNF suspension in K-DS. In the presence of 50 mg/ml of
CNF, all of the tested cells remained alive through-Fig. 1. A photograph of a living Paramecium ingesting CNF. a: At 1 min after the initiation of incubation in a CNF suspension (50 mg/ml), a cell was isolated in 2.5% (w/v) methyl cellulose dissolved in K-DS and mounted on a glass slide.
Then, about 5 min later, a photograph was taken with a digital camera (Nikon, DS-5Mc) attached to a light microscope(ZEISS AX10). A white arrow shows aggregating CNF particles in a food vacuole. b: A photograph was taken at 20 min
after the initiation of incubation in the CNF suspension. Black particles, one of which is shown by a white arrow, indicate
CNF-containing food vacuoles. Bar is 30 µm.

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Cytotoxicity test of nanoparticles using Paramecium

6.25

80

Survival (%)

Su rvi val (% )

100
60
40
20
0
0

24

48

72

12.5

25

50

100
80
60
40
20
0

96

1

10

Tmie of incubation (h)100

CNF concentration (mg/ml)

Fig. 2a. Survival rates of non-proliferating paramecia in a
50 mg/ml CNF suspension. The abscissa shows length oftime of incubation in the CNF suspension, and the ordinateshows the mean percentage of cells surviving up to 96 h
after the initiation of incubation (n=6).

Fig. 2b. Effect of different doses of CNF on survival of
non-proliferating paramecia. The abscissa shows theamount of CNF suspended in K-DS. Scale of abscissa wasshown by logarithm. The ordinate shows the mean percentage of cells surviving up to 96 h after the initiation of incu-

out the period of 96 hours incubation, without any
noticeable detrimental response to CNF exposure(Fig. 2a). No cytotoxicity was detected over arange of CNF concentrations up to 50 mg/ml in
non-proliferating paramecia (Fig. 2b).

culture. A single cell was suspended in 200 µl of
the CNF suspension (50 mg/ml) and then was kept
at 25℃. Under these conditions, during the first 24
hours the paramecia underwent cell division once,but their daughter cells gradually lost the ability toswim and, after 48 hours of incubation, more than
95% died. On the other hand, cells in the control
culture continued cell divisions during 48 hours of
incubation (Fig. 3a).
We examined cytotoxicity effect of CNF at

14

30

12

25

Number of cells

Number of cells

Effect of CNF on proliferation
The effect of CNF on the proliferation of paramecia was tested in fresh culture medium containing nutritive bacteria in a glass depression slide

10
8
6
4

20
15
10
5

2
0

0

0

10

20

30

40

50

0

10

CNF

Fig. 3a. Survival of proliferating paramecia cultured in a
CNF suspension. The abscissa shows time after the initiationof incubation and the ordinate shows the mean number ofsurviving cells in fresh culture medium containing nutritivebacteria and 50 mg/ml CNF (n=6). Control means the culture containing 0 mg/ml of CNF (n=6).

30

40

50

Time of incubation (h)Time of incubation (h)Control

20

0

30

60

120

Fig. 3b. The time course of proliferation in different concentrations of CNF. The mean numbers of cells in fresh
culture medium containing nutritive bacteria are shown(n=6). Diamond, 0; square, 30; triangle, 60; and circle, 120µg/ml CNF. The abscissa shows time after the initiation of
incubation and the ordinate the mean number of living

lower amount of CNF suspension. A single cell
was suspended in 200 µl of culture medium containing bacteria in a glass depression slide culture.
The time course of the CNF effect is shown in Fig.
3b. During the first 24 hours in bacteria-containing
culture medium, CNF did not inhibit proliferation
at any concentration. However, during the second
24 hours incubation, 60 and 120 µg/ml of CNF
inhibited cell division.
The dose effect of CNF on proliferation at 48
hours after incubation is shown in Fig. 4. The
mean number of cells at 30 µg/ml of CNF was
about 80% of that of the control culture (Dunnett,
P<0.05). At 60 µg/ml of CNF, the mean number ofcells decreased to about 40% of the control(Dunnett, P<0.01), and remained at this level as
the concentration of CNF increased. Thus, no dose
effect was observed above 60 µg/ml of CNF.

Number of cells

40
35
30
25
20
15

*
**

**

**

**

10
5
0
0

0.1
0.2
0.3
0.4
CNF concentration (mg/ml)

Number of cells

Jpn. J. Protozool. Vol. 40, No. 2. (2007)

143

40
35
30
25
20
15
10
5
0
0

0.1
0.2
0.3
0.4
CNF concentration (mg/ml)

0.5

Fig. 5. Recovery of proliferative activity after removal of
CNF. The abscissa shows the concentration of CNF and theordinate the mean number of surviving cells at 48 h after
the initiation of incubation. Bars indicate standard deviations (n=6).

48 hours in various CNF concentrations to CNFfree fresh culture medium in a glass depression
slide. As shown in Fig. 5, at 30, 60 and 125 µg/ml
of CNF, the mean number of cells at 48 hours incubation recovered to control levels (CNF: 0 µg/ml). At 250 and 500 µg/ml, the mean number ofcells did not recover fully, although statisticalanalysis showed no significant differences among
the CNF concentrations (Dunnett, P<0.09 at 250µg/ml).

0.5

Fig. 4. Dose effect of CNF on proliferation. The abscissashows the concentration of CNF and the ordinate the mean
number of surviving cells at 48 h after the initiation of incubation. Bars show the standard deviation and ＊ indicates asignificant statistical difference at P<0.05 (Dunnett test) and＊＊ at P<0.01 (Dunnett test) (n=6).

Reversibility of the cytotoxicity effect of CNF
The reversibility of cytotoxicity of CNF was
examined by transferring paramecia cultured for

DISCUSSION
The health risks to humans and other organisms
from exposure to nanoparticles in the environment
are one of the most important concerns regarding
the use of these potentially valuable materials. The
Paramecium bioassay system described here provides a useful means by which to determine thecytotoxicity of environmental and ingested CNF.
Paramecia play an important role in food webs
in aquatic ecosystems. They ingest diverse bacteria
growing in water (Taylor, 1979) and, in turn, are144

Cytotoxicity test of nanoparticles using Paramecium

eaten by other protozoa such as amoebas, didinia
and other metazoan animals or their larvae (Porter
et al, 1979; Taylor, 1980). Thus, CNF could moveinto food webs of aquatic ecosystems following
ingestion by paramecia. The speed of a CNFcontaining food vacuole formation and the maximum number in a cell were similar to those observed after ingestion of nutritive bacteria (data notshown)
Two types of nuclear division occur in Paramecium: mitotic nuclear division performed by the
micronucleus, and amitotic division by the macronucleus. For the purposes of this article, the cells
incubated in K-DS are referred to as "nonproliferating" cells.
At the beginning of this study, we set two categories of cellular properties for an assessment of
the cytotoxicity effect of CNF. One is the propertyof cell survival and the other cell division. For cell
survival tests we used the buffered saline, K-DS,and examined survival ratio and swimming behavior. In non-proliferating paramecia, CNF had nocytotoxicity effect on both cellular properties in all
experiments performed in this study.
However, we have found strong cytotoxic effectof CNF in the cell division tests. In this test we
used culture medium containing lettuce juice andnutritive bacteria as described in “Materials andMethods”. The cytotoxic effect of CNF was observed in both properties of cell survival and cell
division. At 50 mg/ml of CNF, cytotoxic effect was
detected in the property of cell division during theinitial 24-hour incubation and no effect on theproperty of cell survival. In the following 24-hour
incubation, however, cytotoxic effect was detectedin the property of cell survival, too.
We considered the possibility of causal relationof CNF effect on the property of cell survival. The
simplest case is the inhibition of energy source
uptake by CNF. But, it is hard to assume this casebecause, under our experimental conditions, paramecia can survive more than 1 week without any

nutrient biomaterials indicating that the energy
pool of cytoplasm is enough for surviving in the
period of experiments performed in this study.
However, an energy source uptake under the presence of CNF is remained to estimate experimentally.
An alternative possibility is the production of
hazardous chemical substances by interaction between the metabolic products of culture medium
and CNF. At this moment we have no information
about chemical compositions of the culture medium. However, one candidate of the hazardous
chemical substances is suggested by the following
observation. One of the remarkable characteristics
observed regarding cytotoxicity of CNF was the
cessation of ciliary movement on the cell surface
of proliferating paramecia. Ciliary movement inparamecia is essentially controlled by membranepotential (Dunlap, 1977; Kung and Saimi, 1982).
Because the control of membrane potential is dependent on several kinds of ion channels (Kungand Saimi, 1982), chemical substances that affection channel functions will be one of target molecules to understand cytotoxicity of CNF.
Alternative experimental approach to the molecular mechanisms of CNF cytotoxicity is the useof complete synthetic culture medium in a cell
division test. In this experiment we could systematically change chemical compositions in culture
medium, so that the cytotoxicity of CNF control.
As shown in Fig. 4, the dose effect of CNF cytotoxicity was biphasic: at lower concentrations(from 30 to 60 µg/ml of CNF), a dose dependency
was observed, while the effect appeared independent of dose at higher concentrations (from 60 to500 µg/ml of CNF). The number of cells in culture
medium depends on both the number of cell divisions and the number of cell death during cultivation. To obtain precise information about the contribution of cell division and cell death we have toperform a single cell isolation line culture under
the presence of CNF after every cell division inJpn. J. Protozool. Vol. 40, No. 2. (2007)

future experiments.
Our findings indicate that the effect of CNF on
living organisms might not be as simple as previously predicted. We anticipate the presence ofsome complexity involving the molecular interactions between CNF and intracellular chemicals,
which, in turn, may have harmful effects on cells.
In summary, the Paramecium bioassay system
reveals that it is important to distinguish the property of cell division and cell survival in the elucidation of CNF cytotoxicity. As our studies haveindicated, developing the appropriate regulatory
guidelines to ensure the safety of nanotechnology
will require biochemical examination and identification of chemical substances that interact withand modulate CNF cytotoxicity.

ACKNOWLEDGMENTS
Research was performed under Health and Labor Sciences Research Grants for Research on
Risk Assessment of Chemical Substances:“Development of Visualization Method of Dynamical Motion Behavior of Nanoparticles in theInternal Body (H18-Chem-General-006)” from
Ministry of Health, Labor and Welfare of Japan.

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