Download Evaluation of suitable solvents for testing the anti

Indian Journal of Biochemistry & Biophysics
Vol. 47, June 2010, pp. 166-171
Evaluation of suitable solvents for testing the anti-proliferative activity of triclosan
- a hydrophobic drug in cell culture
S Vandhanaa, P R Deepab*, G Aparnac, U Jayanthia and S Krishnakumara*
a
Ocular Pathology, Vision Research Foundation, Sankara Nethralaya, 18, College Road, Nungambakkam, Chennai 600 006, India
b
Biological Sciences, BITS, Pilani-Chennai Centre, India, c Biological Sciences, BITS, Pilani (Rajasthan), India
Received 21 December 2009; revised 16 February 2010
Triclosan, a broad spectrum antibiotic is currently being evaluated for its anti-cancer property. Though several solvents
are available to dissolve lipophilic (hydrophobic) drugs, solubility and toxicity aspects pose a challenge, when combined
with the cell culture medium. In this paper, we present a simple approach based on physico-chemical and biologic criteria to
choose a suitable solubilizing agent to study the anti-proliferative property of triclosan in breast cancer cell line MCF-7.
Triclosan was dissolved in five different solvents viz. DMSO, absolute ethanol, 1 N NaOH, 55% polyethylene glycol + 45%
ethanol mixture (PEM) and acetone and diluted with the culture medium (1 mg/ml). Although triclosan dissolved
completely in all five solvents, on dilution with culture medium, turbidity was observed in DMSO, 1 N NaOH and ethanol.
Cell viability was 95.23% in 10 µl of acetone, when compared with 49.45% at the same volume of PEM. This non-toxic
nature of acetone was supported by DNA fragmentation analysis and phase contrast microscopy. A significant decrease in
cancer cell proliferation at 100 µg/ml of acetone-solubilized triclosan, compared with 100 µg/ml of PEM-solubilized
triclosan (p<0.05) indicated stronger anti-proliferative effect and greater drug-sensitivity of triclosan when solubilized in
acetone. Results showed that acetone-solubilized triclosan was suitable for anti-cancer investigations in cultured MCF-7
cells.
Keywords: Triclosan, Cytotoxicity, Cell culture, Drug sensitivity, Acetone, Polyethylene glycol
Triclosan, a broad spectrum antimicrobial agent has
also been widely used for the last three decades in
soaps, deodorants, cosmetics and toothpastes1,2. It acts
as antibacterial agent by inhibiting the enoyl reductase
activity of the type II fatty acid synthase in bacteria.
Zuckerbraun et al.3 studied the in vitro cytotoxicology
of triclosan, the active ingredient in some mouth
rinses and dentifrices used in the prevention and
treatment of gingivitis and plaque using the in vitro
cell culture system of Smulow-Glickman (S-G)
human gingival epithelial cell line. Triclosan is also
being investigated for its anti-cancer properties due to
its inhibition of type I fatty acid synthase, a potential
chemotherapeutic target for cancer4.
Since triclosan is insoluble in water, it needs to be
solubilized in organic solvents or other formulations to
enable its bioavailability. However, its biological
activities such as anti-plaque and anti-inflammatory
______________
*Authors for correspondence
Tel: 91-44-28271616, Extn- 1302; Fax: 91-44-28254180
E-mail: [email protected], [email protected]
Abbreviations: DMSO, dimethyl sulphoxide; MTT, 3-(4, 5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PEM,
55% polyethylene glycol + 45% ethanol mixture.
effects are lost, when unsuitable solubilizers are used5, 6.
Kjaerheim et al5 experimented the dissolution of
triclosan in different oils (olive, soy, and sunflower
oils), as well as in polyethylene glycol (PEG) and
glycerol, wherein they found that triclosan dissolved
in oils lost its anti-bacterial activity in vitro, though
the oils in themselves exhibit significant plaque
inhibition. The results of their study was of
significance in the context of toothpastes and mouth
rinses that contain flavoring oils and occasionally also
glycerol and PEG, wherein these substances are likely
to interfere with the clinical effect of triclosan in these
products7. In another study8, it was shown that the
nature of the solubilizing agent influenced the antioral malodour activity of triclosan.
In order to investigate the anti-proliferative effect
of triclosan in cancer cells grown in vitro, in this
study, we have evaluated the role of the solvent in
combination with the specific cell culture medium
using a breast cancer cell line (MCF 7 cells). Earlier,
the solvents such as DMSO, ethanol, alkali, acetone,
and 55% polyethylene glycol-400 + 45% ethanol
mixture (PEM) have been used for dissolving
triclosan4,6,9. However, in the context of cell culture-
VANDHANA et al: SUITABLE SOLVENT FOR TESTING TRICLOSAN IN CELL CULTURE
based testing of anti-proliferative (cytotoxic) property
of triclosan, no systematic evaluation of solvents has
been carried out, so far. We present a simple approach
to select a suitable solubilizing agent that would
enable testing of a lipophilic drug’s anti-proliferative
activity in cells grown in vitro.
Materials and Methods
Materials
Triclosan (2, 4, 4’-trichloro-2’-hydroxydiphenyl
ether), MTT [3-(4, 5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide], dimethyl sulphoxide
(DMSO) were obtained from Sigma-Aldrich, USA.
Polyethylene glycol 400 (PEG-400) was obtained
from Hi Media (Mumbai, India) and other chemicals
were purchased from Merck (Mumbai, India).
Cell lines and culture conditions
Breast (mammary) cancer cell line MCF-7 was
obtained from National Centre for Cell Science
(NCCS), Pune, India. The cells were maintained in
DMEM supplemented with 750 µl/dl calf insulin
(40 IU/ml) and 10% fetal bovine serum at 37ºC in an
atmosphere with 5% CO2.
Test of solubility and pH
Triclosan was dissolved in five different solvents:
(i) DMSO, (ii) acetone, (iii) 55% PEG-400 + 45%
ethanol mixture (PEM), (iv) absolute ethanol, and
(v) 1 N NaOH, and then diluted with the culture
medium to obtain a final concentration of 1 mg/ml.
The drug was considered completely solubilized when
it formed a clear solution in its solvent and produced
no turbidity or precipitate, when diluted with the
medium. Any precipitation or turbidity observed
visually in the final solution containing the drug +
solvent + medium was recorded. The pH was
measured with a pH meter (LI120-ELICO India).
Cell viability (MTT) assay
MCF-7 cells were seeded at 2.5 × 103 cells/well in
96-well plates with 100 µl of culture media and
incubated at 37°C for 24 h and then fresh medium
was added. Triclosan dissolved in acetone and PEM
(selected after the above-mentioned preliminary
screening of five different solvents) was added at a
range of concentrations from 50-200 µg/ml to the
wells. The solvent controls contained the
corresponding volumes (5-20 µl) of solvent alone,
without the drug. The cells were then incubated for
48 h at 37°C. Thereafter, the medium was removed
167
and 100 µl of fresh medium and 10 µl of MTT
(5 mg/ml) was added to all the wells and incubated
for 4 h at 37°C. The supernatant was then removed
and 100 µl DMSO was added to dissolve the
formazan crystals. Color developed was read at
570 nm. Cell viability (or cell survival) was calculated
as: (Test OD/Control OD) × 100. The 50% inhibitory
concentration (IC50) of triclosan solubilized in acetone
and in PEM was calculated using polynomial
regression analysis using Microsoft Excel.
Assessment of cell morphology by phase contrast microscopy
MCF-7 cells treated with PEM and acetone were
assessed for morphological changes using a phase
contrast microscope (Nikon). The cells were exposed
to these solvents in volumes corresponding to the
volume required to dissolve triclosan at its 50%
inhibitory concentration IC50.
DNA fragmentation assay (Agarose gel electrophoresis)
MCF-7 cells were plated at 2.5 × 104 cells/well in
12-well plates and incubated for 24 h. Then the
medium was removed and fresh medium and
respective solvent controls (5-20 µl) were added to
the wells and incubated for 48 h. At the end of
incubation, the cells were collected and pelleted and
the DNA was extracted by Qiagen (Germany) kit as
per the manufacturer’s instructions. The extracted
DNA was electrophoresed at 100 V in a 2% agarose
gel with 0.5% ethidium bromide for 2 h. The obtained
DNA fragments were visualized under UV light and
documented with a gel documentation system
(Vilber Lourmat, France). The DNA from solventincubated cells were run along with that of untreated
MCF-7 cells (control), toxic H2O2-treated cells as
positive control for DNA laddering, and a
100 bp marker.
Statistical analysis
The results were expressed as mean ± SD of values
obtained from a minimum of three independent
experiments, each performed in triplicate. In all the
experiments, the untreated cells with the culture
medium served as the control. Comparisons between
specific groups as mentioned in the results section
were done using student’s t-test.
Results
Solubility of triclosan
Differences in triclosan solubility were observed in
the five solvents made up with the culture medium
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INDIAN J. BIOCHEM. BIOPHYS., VOL. 47, JUNE 2010
(final conc: 1 mg/ml) (Fig. 1). Triclosan did not
dissolve completely in DMSO (bottle 1), absolute
ethanol (bottle 2) and 1 N NaOH (bottle 3) when
diluted with culture medium, resulting in a turbid
solution. However, PEM (bottle 4), and acetone
(bottle 5) solubilized triclosan diluted with the culture
medium resulted in a clear solution. pH was also
recorded in these triclosan + solvent + culture
medium solutions. The pH of the final solutions with
1 N NaOH and absolute ethanol was unfavorable
(pH 8.0) for cell culture conditions while with
acetone, PEM and DMSO an optimal pH of 7.4 was
obtained. Based on the solubility and pH criteria, only
two solvents (acetone and PEM) were selected for
further evaluation of suitability as triclosan
solubilizers in cell culture conditions.
Effect of solvents on cultured cells
Toxicity of the solvents (5, 7.5, 10 and 20 µl) was
evaluated in terms of percentage viability of MCF-7
cells incubated with the solvents only. Fig. 2
Fig. 1—Bottles 1 to 5 represent triclosan dissolved in DMSO,
absolute ethanol, 1 N NaOH, PEM, and acetone respectively, and
made up with the culture medium. Triclosan did not dissolve
completely in the solvents in bottles 1 through 3 on addition of the
culture medium giving a turbid appearance. However, PEM
(bottle 4), and acetone (bottle 5) solubilized triclosan diluted with
the culture medium resulted in a clear solution.
Fig. 2—Effect of acetone and PEM on cell viability presented as
percentage of control (cells with medium only) [Cells were grown
for 48 h at various concentrations of the solvents. Values are
expressed as the mean ± S.D. of three separate experiments in
triplicate. Differences in cell viability corresponding to acetone
and PEM solvents in each concentration were compared by
student’s t-test. * represents significant difference at p<0.05]
represents MTT assay results of acetone and PEM,
respectively. A significant decrease was observed in
the viability of cells incubated with PEM, in
comparison with those incubated with acetone
(p<0.05). At the same volume (10 µl), while acetoneincubated cells had 95.23% viable cells, PEMincubated cells had only 49.45% viable cells, strongly
indicating the cytotoxicity of PEM in MCF-7 cell
culture conditions.
Cell morphology analysis (Fig. 3c) showed
shrinkage of cells with PEM in MCF-7 cells, while
there was no observable change in the morphology
of cells incubated with acetone (Fig. 3b).
Further, agarose gel electrophoresis revealed mild
oligonucleosomal DNA fragmentation (Fig. 4) with
PEM, suggesting its cytotoxicity. On the other hand,
acetone-incubated group was comparable with the
untreated control (Fig. 5).
Fig. 3—Effect of solvents on MCF-7 cell morphology observed
by phase contrast microscopy. [(a): untreated MCF-7 cells;
(b): MCF-7 cells exposed to acetone alone (volume corresponding
to dissolution of triclosan at its IC50) showed no observable
morphological changes; and (c): MCF-7 cells exposed to PEM
alone (volume corresponding to dissolution of triclosan at its IC50)
revealed shrinkage of cells]
Fig. 4—Cytotoxic effect of PEM on MCF-7 cells analyzed by
DNA fragmentation assay [Lane 1, control untreated MCF 7 cells;
and lanes 2 to 5, solvent volumes 5, 7.5, 10, and 12.5 µl
respectively. Cells treated with different volumes of PEM
revealed fragmented DNA that can be compared with the positive
control served by H2O2 treated cells (lane 6), and a standard
marker DNA ladder (lane 7) is shown along side]
VANDHANA et al: SUITABLE SOLVENT FOR TESTING TRICLOSAN IN CELL CULTURE
Fig. 5—DNA fragmentation assay on acetone solvent incubated
MCF-7 cells [Lane 1, control untreated MCF 7 cells; lanes 2 to 4,
solvent volumes 5, 7.5, and 10 µl respectively; lane 5, positive
control served by H2O2 treated cells; and lane 6, a standard marker
DNA ladder. The DNA corresponding to different acetone
volumes are comparable with the control untreated MCF-7 cells]
Effect of solvents on anti-proliferative activity of triclosan
The anti-proliferative effect of triclosan was
assessed, when solubilized in acetone and PEM by
MTT assay (Fig. 6). Panels of triclosan concentrations
between 50-200 µg/ml were chosen. The IC50
(50% inhibitory concentration of the drug over cell
viability) was obtained by interpolation. IC50 of
triclosan solubilized in acetone was found to be
91.09 µg/ml, while in PEM, it was 121.25 µg/ml. This
implied that a 1.33-fold increase in drug concentration
was required to induce anti-proliferative effects in the
cells, when triclosan was dissolved in PEM in
comparison
with acetone-dissolved
triclosan,
indicating lesser drug-sensitivity in PEM. A
significant decrease in cell proliferation at 100 µg/ml
of acetone-solubilized triclosan, compared with
100 µg/ml of PEM-solubilized triclosan (p<0.05) was
observed, indicating a stronger anti-proliferative
effect and greater drug sensitivity of triclosan when
solubilized in acetone.
Discussion
Triclosan has been known for its broad-spectrum
anti-microbial activity for nearly three decades10 and is
also reported to inhibit the mediators of inflammation,
thereby exhibiting anti-inflammatory effects11. It is
now being investigated for its anti-proliferative activity
and seems to have promising anti-cancer effects12.
Being a lipophilic organic molecule, solubilizing
triclosan in a solvent, followed by culture medium
posed some difficulties in testing triclosan’s antiproliferative activity in cells grown in vitro. Here, we
169
Fig. 6—Inhibitory effect of triclosan solubilized in acetone and
PEM differ, as observed by MTT assay in MCF-7 cells incubated
with triclosan for 48 h [Difference in cell viability corresponding
to each concentration (50, 75, 100 and 200 µg/ml) of triclosan
solubilized in acetone and triclosan in PEM was compared by
student’s t-test. * represents significant difference at p<0.05.
Triclosan dissolved in acetone showed significantly lower
percentage of cell viability than PEM-dissolved triclosan at
100 µg/ml concentration (p<0.05). Further, the IC50 value of
PEM-solubilized triclosan was about 1.33-fold higher than the
IC50 of acetone-solubilized triclosan (121.25 µg/ml and 91.09
µg/ml respectively)]
describe a systematic approach for deciding a solvent
that not only dissolves the hydrophobic drug, but is
also compatible biologically for experimentation with
triclosan on cells grown in vitro.
Triclosan is an important anti-microbial constituent
in personal care and oral care products. Being
sparingly soluble in water, product formulations take
into account the solubilizers that are effective in not
only producing stable formulations, but also
maximize the anti-bacterial efficacy of triclosan in the
formulation. In this context, Taylor et al.13 suggested
formulation approaches, wherein they clearly
demonstrated
an
association
between
the
antimicrobial activity and the physico-chemical
characteristics of triclosan in aqueous solution,
solution with added co-solvent (propylene glycol) and
surfactant solutions (sodium lauryl sulphate and
ammonium lauryl sulphate)13. We found that although
triclosan was completely soluble in DMSO, NaOH
and ethanol, dilution with culture medium ideal for
growing MCF-7 cells resulted in turbidity or
unfavorable pH change that was not conducive for
further testing in cultured cells. On the other hand,
both in acetone and PEM a clear solution of
solubilized triclosan was obtained in culture medium.
However, we further sought to verify amongst these
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INDIAN J. BIOCHEM. BIOPHYS., VOL. 47, JUNE 2010
two solvents, if any of them interfered with the
biologic function of triclosan in inhibiting
proliferation of MCF-7 cells in vitro.
Our results clearly indicated that acetonesolubilized triclosan was suitable for testing the drug
in MCF-7 cells grown in vitro. Acetone was shown to
be a non-cytotoxic solvent (an average of 97% cell
viability in the solvent in volume ranging from
5-20 µl). This was in contrast with the cytotoxicity of
PEM solvent on MCF-7 cells with an average cell
viability of 58% in the selected range of solvent
volumes (5-20 µl). Acetone-treated cells showed
intact DNA, while PEM incubated MCF-7 cells
showed DNA fragmentation on agarose gel
electrophoresis, suggestive of its toxicity on MCF-7
cells.
The studies6, particularly in dental research have
investigated the role of solvents in determining the
biological efficacy of triclosan. In a study by
Kjaerheim et al.5, it has been reported that triclosan
alone, dissolved in a suitable solvent has an
antiplaque effect and that the nature of detergent or
organic solvent used to dissolve triclosan affects its
clinical effect.
The IC50 of triclosan in acetone on MCF-7 cells
was 91.09 µg/ml, while that of triclosan solubilized in
PEM showed a rise by 1.33-fold to 121.25 µg/ml. The
higher IC50 of triclosan in PEM implied a decreased
drug-sensitivity in PEM, which might be due to the
large molecular size of PEG-400 known to form
micelle-like structures. These may bind with the
triclosan molecule, thus rendering it less available for
biological activity6. The marked reduction in cell
viability at 100 µg/ml (p<0.05) on treatment with
acetone-solubilized triclosan in comparison with
PEM-solubilized triclosan clearly indicated greater
biological activity of triclosan dissolved in acetone in
countering the proliferation of MCF-7 cells grown
in vitro.
Testing water-insoluble drugs in cells grown
in vitro often requires organic solvents to solubilize
the drug in culture medium that may result in toxicity
on the cells and may interfere with the biological
activity of the test-drug14. Factors, such as the solvent
volume required to solubilize the drug, duration of
incubation with the cells and the specific chemistry of
the solvent and drug (alone and in combination)
would determine the choice of solvent. Further, the
suitability of solvent with respect to the specific type
of cell line needs to be evaluated. For instance, Pace
and Elliott15 found that various cell lines grown
in vitro showed differences in their degree of
sensitivity to different chemicals such as acetone and
phenol. They found that the skin cells are more
sensitive to acetone’s effects than the fibroblast cells,
wherein a concentration of 5.0 mg/ml is not toxic to
fibroblasts within a 10-day period, although it is
noticeably toxic to the skin cells.
To conclude, acetone demonstrated favorable
physico-chemical and biologic characteristics to
solubilize triclosan and to study its anti-proliferative
effect in a breast cancer cell line. This step-wise
approach of selecting a solubilizing agent can also be
extended to other hydrophobic drugs for evaluation of
their anti-proliferative potential in cultured cells. The
results of this study bring out different aspects of a
solvent-cell culture system emphasizing on criteria
such as solubility, pH, solvent toxicity, and
interference of solvent with drug efficacy. With the
advent of newer pharmaceutical solvents, such as
room temperature ionic liquids (green solvents) that
greatly increase the solubility of water-insoluble
drugs, their effects in cell culture system would need
careful and systematic examination16,17.
Acknowledgement
The authors acknowledge the project grant
(No. 58/15/2005-BMS) from the Indian Council of
Medical Research, New Delhi. This work in part was
presented at the 12th International Conference of the
Indian Society of Chemists and Biologists
(February, 2008).
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