DRUG DEVELOPMENT RESEARCH •• : ••–•• (2012)

DDR
Research Article

Cytotoxicity of a Novel Oil/Water Microemulsion

System Loaded with Mitomycin-C in In Vitro Lung
Cancer Models
Mustafa Kotmakchiev,1,2 Gülten Kantarcı,1* Vildan B. Çetintaş,3 and Gökhan Ertan2
1
Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Ege University, Bornova,
Izmir 35100, Turkey
2
Department of Pharmaceutical Technology, Faculty of Pharmacy, Ege University, Bornova,
Izmir 35100, Turkey
3
Department of Medical Biology, Faculty of Medicine, Ege University, Bornova, Izmir 35100,
Turkey

Strategy, Management and Health Policy

Enabling Preclinical Development Clinical Development

Technology, Preclinical Toxicology, Formulation Phases I-III Postmarketing
Genomics, Research Drug Delivery, Regulatory, Quality, Phase IV
Proteomics Pharmacokinetics Manufacturing

ABSTRACT The aim of the study was to develop a novel oil/water microemulsion system to increase the
cytotoxic effect of mitomycin C (MMC) on human lung cancer cell lines through comparison to the
conventional MMC solution. The microemulsion formulation was composed of soybean oil, lecithin, Tween
80, ethanol, and water. Characterization of the microemulsions was carried out by means of particle size,
viscosity, conductivity, storage stability, in vitro drug release, and in vitro hemolysis. Putative anticancer
activity was determined using Calu1 and A549 carcinoma cell lines with an XTT [2,3-bis-(2-methoxy-4-
nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] cell proliferation assay. Drug release from the MMC-
loaded microemulsion was sustained for more than 5 h while release from MMC solution was completed
within 2 h. Based on the results of cytotoxicity study, a higher anticancer effect was observed with mitomycin
C-loaded microemulsion (MMC-M), with IC50 values being approximately twofold to fourfold higher than
that seen with the MMC solution on Calu1 and A549 carcinoma cell lines, respectively. In conclusion, MMC
microemulsion has several advantages including slower drug release, a more pronounced anticancer effect
at lower MMC doses, potentially leading to lower systemic toxicity potential if leakage occurs from the tumor
site. Drug Dev Res •• : ••–••, 2012. © 2012 Wiley Periodicals, Inc.

Key words: microemulsion; mitomycin C; Calu1; A549

INTRODUCTION combination with other anticancer agents to treat col-

Mitomycin C (MMC) is a potent antineoplastic
antibiotic that is naturally synthesized by Streptomyces
caespitosus. It requires activation by reductive enzymes
under hypoxic conditions to produce its anticancer
effects [Iyer and Szybalski, 1964; Bligh et al., 1990].
When activated, MMC forms cross-links between
neighboring guanine bases on the complementary deox-
yribonucleic acid (DNA) strands [Tomasz et al., 1987],
forms free radicals that contribute to the elimination of
cancer cells [Emanuel et al., 1984], and induces apop-
tosis [Pirnia et al., 2002]. It is used as a single drug or in
orectal and anal cancer, bladder cancer, cervix cancer,
gastric cancer, lung cancer, pancreas cancer, etc.
*Correspondence to: Gülten Kantarcı, Department of
Pharmaceutical Biotechnology, Faculty of Pharmacy, Ege
University, Bornova, Izmir 35100, Turkey.
E-mail: gulten.kantarci@ege.edu.tr
Received 27 January 2012; Accepted 20 February 2012
Published online in Wiley Online Library (wileyonlinelibrary.
com). DOI: 10.1002/ddr.21007

© 2012 Wiley Periodicals, Inc.

2 KOTMAKCHIEV ET AL.
[Bradner, 2001]. However, systemic administration of
this drug is associated with severe side effects, including
bone marrow depression and renal and gastrointestinal
damage [Allan et al., 1993]. Furthermore, the biological
half-life of MMC is short, approximately 50 min [Den
Hartigh et al., 1983]. In order to overcome these side
effects of MMC, and to obtain higher treatment efficacy,
many attempts have been made to incorporate the drug
in different delivery systems. These include intratumoral
injection of MMC–dextran conjugates [Nomura et al.,
1998], arterial chemoembolization with polyvinylalcohol
or degradable starch microspheres [Rump et al., 2002;
Shitara et al., 2008], and incorporation of MMC in poly-
lactic acid nanoparticles [Hou et al., 2009].
Intratumoral injection of antineoplastic drugs
can provide highly localized drug concentrations with
greatly reduced systemic toxicity [Almond et al., 2003].
Injection of anticancer drugs directly into the endo-
bronchial tumor or infiltrated mucosa via a fiber optic
bronchoscope, reduced tumor mass with the accom-
panying atelectasis, or obstructive pneumonia being
improved [Wagai et al., 1981]. Çelikoğlu et al. [1997,
2006] also used direct intratumoral injection of mito-
mycin, cisplatin, 5-fluorourasil, methotrexate, bleomy-
cin and mitoxantrone into the endobronchial tumors
and found that endoscopically visible tumors were
reduced in size, and infiltrative changes were also
improved. The therapy was well-tolerated with no sys-
temic side effects or serious complications.
Several microemulsion (M) systems have also
been developed for intratumoral delivery where the
cytotoxic effects of incorporated anticancer drugs are
enhanced [Karasulu et al., 2004, 2009; Shiokawa et al.,
2005].
Ms are self-aggregated systems in which oil and
water are homogenously mixed in the presence of
amphiphiles. They differ from conventional emulsions
not only in their ease of preparation and much smaller
structural size but also in their thermodynamic stability,
which allow for a long-lived stabilization of mixed oil/
water (o/w) systems [Garadzielsky, 2008]. The obvious
benefits of Ms as drug delivery systems have led to the
development of long-term stable systems for parenteral
administration [Lv et al., 2005; Date and Nagarsenker,
2008].
The development of a carrier system was planned
in this study to increase the magnitude of antitumor
effect of MMC in endobronchial applications without
increasing systemic side effects. For this purpose, an
o/w M with appropriate viscosity and pH suitable for
application to endobronchial tumors via a fiber optic
bronchoscope was developed; MMC was loaded in the
M, and the antitumor effect of the system on human
lung cancer cell lines were investigated in vitro.
Drug Dev. Res.
MATERIALS AND METHODS
Materials
Soybean oil was donated by Orkide Oils Inc.
(Izmir, Turkey). MMC was purchased from Onko-
Kocsel Co. (Istanbul, Turkey). Soybean lecithin (L)
(90% phosphatidylcholine) was obtained from Appli-
Chem GmbH (Darmstadt, Germany), Tween 80 (T80)
originated from Merck-Co. (Hohenbrunn, Germany),
ethanol (E) (assay ⱖ 99.9%), methanol, and potassium
dihydrogen phosphate were from Merck KGaA (Darm-
stadt, Germany), disodium hydrogen phosphate from
Carlo Erba Reagenti (Milano, Italy), sodium chloride
was from Atabay (Istanbul, Turkey), and dialysis bag
(Spectra/por dialysis membrane, M.W. cutoff 12,000–
14,000, 32 mm ¥ 104 mm flat width by flat length,
volume length: 3.3 ml/cm) from Spectrum Laboratories
Inc. (Rancho Dominguez, CA). Cell culture media
Ham’s F12 and Dulbecco’s modified Eagle’s medium
(DMEM) as well as fetal bovine serum and penicillin
and streptomycin were from Biological Industries (Beit
HaEmek, Israel), and the XTT cell proliferation kit was
purchased from Roche Diagnostics (Istanbul, Turkey).
Methods
Octanol/water partition coefficient determination
A stock solution of MMC (1 mg/ml) was prepared
and dispensed into Eppendorf tubes (Corning Inc.,
Lowell, CA; 1 ml/tube). An aliquot (1 ml) of n-octanol,
previously saturated with distilled water for 24 h was
added, and the tubes were shaken at 25 ⫾ 1°C and
125 rpm for 3 h and left for equilibration overnight.
The absorbance of the water phase was measured spec-
trophotometrically at 362 nm and the amount of the
drug diffused into the oil phase after the equilibration
was calculated.
Preparation of oil-in-water M
For M formulation, soybean oil was used as the oil
phase. L and T80 were used as surfactants and ethanol
was cosurfactant. L was dissolved in a predetermined
amount of ethanol by stirring with a vortex mixer at
1,000 rpm. Oil and T80 were then added and mixed.
Finally, double distilled water was added to the oily
mixture drop by drop in increasing amounts by stirring
with a magnetic stirrer (IKA Labortechnik, Staufen,
Germany) at room temperature as dilution series. The
systems were then observed visually for transparency.
Clear and transparent monophasic mixtures were con-
sidered to represent viable M systems. In preliminary
studies, the capability of the M formation was investi-
gated by varying the surfactant–cosurfactant mixture
(S/coS) ratio (2:1, 1.5:1, 1:1) and the T80/L ratio (4:1,
 IN VITRO CYTOTOXICITY OF MITOMYCIN C MICROEMULSION
3:1, 2:1, 1:1). Following this, the phase diagrams were
constructed with different S/CoS and T80/L ratios.
Ideal S/coS weight ratios and micremulsion existence
areas were determined with the aid of phase diagrams
drawn using a computer program developed in the
computer center at the Ege University Faculty of Phar-
macy [Ege et al., 2004]. At the last step, MMC was
added to the M formulation (1 mg/g).
Characterization of Ms
Refractive indices were measured with a digital
liquid refractometer with thermometer (Atago NAR-1T,
Tokyo, Japan) at 25.7 ⫾ 0.6°C. The electrical conductiv-
ity was measured using a conductometer (Jenway 4071;
Bibby Scientific Ltd., Stafford, UK) at 20 ⫾ 2°C. Viscos-
ity measurements were carried out at 25.0 ⫾ 0.1°C
using a Brookfield Programmable DV-III+ Rheometer
(Brookfield Engineering Labs, Inc., Middleboro, MA)
(Spindle: SC4-21). Measurements were made on a Zeta-
sizer Nano ZS instrument (Malvern Instruments Ltd.,
Worcestershire, UK) at 25°C. The instrument contains a
4-mW He-Ne laser operating at a wavelength of 633 nm
and incorporates noninvasive backscatter optics. Mea-
surements were made at a detection angle of 173° and
the measurement position within the cuvette was auto-
matically determined by software. Droplet size mea-
surements were performed both for newly prepared and
stored samples. The observed pH values of the samples
were measured by Inolab level 1 pH meter (WTW
Wissenschaftlich-Techniche Werkstätten GmbH, Weil-
heim, Germany) at 20 ⫾ 2°C.
Stability study
The long-term stability of the formulations was
evaluated by visual inspection on a daily basis over a
period of 8 months. The maintenance of clarity and lack
of phase separation were the indicators for the M sta-
bility. Furthermore, the physical stability of the Ms was
assessed by centrifugation method. Samples of Ms
equilibrated for 24 h were subjected to centrifugation
5,000 rpm for 15 min and visually inspected for clarity.
Formulations with no phase separation or no turbidity
formation were considered stable. In order to investi-
gate the stability during temperature changes, Ms were
frozen at -20°C for 30 min and thawed at 40°C for
15 min. This cycle was repeated three times, the
appearance of the formulations was checked, and those
with no phase separation or no turbidity formation were
accepted as stable. Particle size measurement was used
in determining the storage stability of both MMC
loaded and unloaded Ms.
3
In vitro release study
Drug release was conducted using a dialysis tech-
nique. Dialysis bags (Spectra/por, M.W. cutoff 12,000–
14,000, volume length: 3.3 ml/cm) were hydrated with
distilled water for 5 min and approximately 2 g of the
formulation was transferred into the bags. Both ends of
the bag were clamped with Spectra/por closures, one of
which was magnetic weighted and immersed in a beaker
containing 150 ml of normal saline. The experiment was
maintained at 37°C by stirring at 300 rpm. Samples
(3 ml) were withdrawn at predetermined times, placed
to the release medium immediately and analyzed for
drug concentration spectrophotometrically at 362 nm.
Three replicates of each experiment were performed.
Hemolytic Potential
Red blood cell (RBC) preparation
RBC suspension was prepared for hemolysis test
as follows. Human erythrocytes were isolated from
fresh heparinized blood immediately after withdrawal
from a volunteer. The blood was centrifuged at
3,000 rpm for 10 min. The obtained erythrocytes were
separated from the upper serum layer and washed with
phosphate buffered saline (PBS) (pH 7.4) in order to
remove white blood cells and other debris before cen-
trifugation at 5,000 rpm for 2 min. This process was
repeated three times. After the final washing and cen-
trifugation step, packed RBCs were resuspended in
PBS to a hematocrit level of approximately 50%.
Hemolysis test
In order to determine the hemolysis caused by
the interaction between RBCs and the unloaded and
MMC-loaded M formulations, the modified static
method of Reed and Yalkowsky [1985] in the next
section was used. In addition, the surfactants and MMC
solution at the same concentration as in the M formu-
lation were also examined for hemolytic activity. Briefly,
a volume of 100 ml RBC was introduced to Eppendorf
tubes, then formulations at 0.01, 0.05, 0.1, and 0.2 were
added and gently mixed with a pipette and left for
incubation at 37°C for 30 min. The volume of each tube
was then adjusted to 1 ml with PBS and the tubes were
shaken until the formulation–RBC mixture was com-
pletely dispersed. Finally, the tubes were centrifuged at
5,000 rpm for 2 min. Aliquots of 100 ml were withdrawn
from the supernatant and diluted to 10 ml with distilled
water. The absorbance of hemoglobin released from
the lysed RBCs was measured spectrophotometrically
at 414 nm with a double beam spectrophotometer (Shi-
matzu Corp., Kyoto, Japan; UV-1601). Distilled water
was used as blank. The 100% hemolysis level was
defined as the absorbance of hemoglobin at 414 nm in
Drug Dev. Res.
4 KOTMAKCHIEV ET AL.

the supernatant after complete hemolysis of erythro-

TABLE 1. Different Combinations of Microemulsion Components
cytes with distilled water at 37°C (100 ml RBC/10 ml Tested for In Vitro Toxicity
H2O). As the negative control, erythrocytes were incu-
bated in PBS at 37°C. The hemolysis percentage Different Combinations of the contents
obtained was calculated with equation (1). Component % A B C D E F G H M5

H% = ( A − A 0 ) × 100 / ( A100 − A 0 ) (1) Soybean oil 3.4 + + + +

where A is the absorbance value of the test supernatant
and A0 and A100 are absorbance values of the negative
and positive control, respectively. In the cases where a
negative control did not produce any absorbance, A0
was excluded from the equation. All experiments were
performed in triplicate.
Cytotoxicity Study
Cell lines and culture conditions
Human nonsmall cell lung cancer adenocarci-
noma A549 cells were cultured in Ham’s F12 and
epidermoid carcinoma Calu1 cells were cultured in
DMEM medium; both cell lines were obtained from
the American Type Culture Collection (Manassas, VA)
and grown in a highly humidified atmosphere of 5%
CO2 at 37°C. Each medium was supplemented with
10% fetal bovine serum, 100 U/ml penicillin, and
100 mg/ml streptomycin.
In vitro antitumor activity
In order to determine antitumor activity of the
empty M, MMC-M, and MMC solution, cell viability
was evaluated using the XTT colorimetric assay kit in the
presence of different dilution rates of the mentioned
formulations. Briefly, 1 ¥ 105 cells were inoculated onto
each well of a 96-well plate and incubated for 24 h at
37°C in the CO2 incubator. When the cells were attached
on 96-well culture plates, the culture medium was
replaced with fresh DMEM containing increasing con-
centrations of MMC-M, MMC-sol (3–300 mM), or
empty M (0.1–10 ml/well). Empty M was applied at the
same volume of MMC loaded M for the evaluation of its
cytotoxicity on cells. The cells were incubated again at
37°C for 24 h. XTT reagent was added to the wells.
Formazan formation was quantified spectrophotometri-
cally at 450 nm using a microplate reader (Thermo,
Vantaa, Finland) after a 4-h incubation of the plates with
XTT at 37°C in an atmosphere of 5% CO2 + 95% air. All
determinations were confirmed using at least three rep-
licates of each experiment. Untreated cells were evalu-
ated as control. IC50 values were calculated by nonlinear
regression analysis (GraphPad Prism version 5.01,
GraphPad Software Inc., San Diego, CA) comparing the
logarithmic MMC concentrations and the absorbance
values that were revealed by the XTT assay.
Lecithin 8.3 + + + + + +
Tween 80 16.7 + + + + +
Ethanol 16.7 + + + + + +
NaCl 2.4% 2.4 + + + + + + + + +
Water (q.s.) 100 + + + + + + + + +
Potential cytotoxicity of M components
Each component of the M was tested for potential
cytotoxic effects. For this purpose, a simple solution or
various combinations of the M components were pre-
pared with ultrapure distilled water (Table 1) and
tested for cytotoxicity on Calu1 cell culture for 24 h at
3 ml/well. The concentration of each component was
the same as that in the M formulation.
Statistical analyses
The results of the release study were analyzed by
repeated-measures analysis of variance (ANOVA) at
P < 0.05 level of significance. Results of the in vitro
anticancer activity study were analyzed through facto-
rial ANOVA (3 ¥ 8). The interaction between the for-
mulations (3) and the drug doses (8) was determined as
significant; therefore, one-way ANOVA was applied
next, followed by post hoc Tuckey B-test at a signifi-
cance level of P < 0.05. The results of the cytotoxicity
test of different mixtures of the M components were
compared with the control by one-way ANOVA fol-
lowed by post hoc Dunnett t test.
RESULTS
M Preparation and Characterization
In preliminary studies, the capability of the M
formation was investigated by varying the S/coS ratio
(2:1, 1.5, 1:1) and the T80/L ratio (4:1, 3:1, 2:1, 1:1).
Following this, the phase diagrams were constructed
with different S/CoS and T80/L ratios. Then, ideal
T80/L ratio and S/CoS ratio were found as 2:1 and 1.5:1,
respectively, because of forming stable monophasic
systems and larger M formation area. Figure 1 shows
o/w formation region in phase diagram with ideal T80/L
ratio and S/CoS ratio. Five stable and transparent M
formulations were selected in o/w M area for physico-
chemical characterization studies such as pH, viscosity,
droplet size, and conductivity measurements (Table 2).

Drug Dev. Res.

 IN VITRO CYTOTOXICITY OF MITOMYCIN C MICROEMULSION 5

The results of characterization studies are shown in Stability Study

Table 3. For further studies, M5 formulation was
selected for drug loading owing to its lower viscosity,
smaller droplet size, lower polydispersity index (PDI)
value and lower S/Cos content. As seen in Table 3, after
loading MMC into the M, droplet size was found larger
than unloaded formulation (M).
The centrifuge test showed no turbidity or phase
separation for any of the formulations, indicating that
they were all stable [Djordjevic et al., 2005; Cho et al.,
2008]. The formulations also remained stable after the
temperature test, which indicated that they were stable
under conditions of different temperature (–20/+40).
Long-term stability study showed that the selected for-
mulations (M1–M5) had no flocculation, phase separa-
tion, or turbidity at the ambient storage conditions over
a period of 8 months.
The storage stability of the M with or without drug
was checked by observing droplet size values at the end
of predetermined time intervals for 2 months. Figure 2
shows the changes in droplet size of empty (M5) and
drug-loaded M (MMC-M).
After 20 days of storage, a decrease was observed
in droplet size for both of M5 and MMC-M. After the
20th day, the empty M exhibited no significant changes
in terms of droplet size during 2 months. The droplet
size of MMC-M was enlarged to 198 nm, and after-
wards, at the end of 2 months, no significant change was
determined.

In Vitro Release Study

Fig. 1. Pseudoternary phase diagram of the microemulsion system.
The gray area represents the clear transparent microemulsion systems.
The white area indicated with a broken line represents systems that are
turbid two phase mixtures. 䊊, formulations selected for physicochemi-
cal characterization, •, formulation selected for drug loading. S Oil,
soybean oil, S/coS, surfactant–cosurfactant mixture.
As shown in Figure 3, MMC release from the M
was slower compared with that from aqueous MMC
solution. The drug release from MMC-M continued for
more than 5 h, while the release from the aqueous solu-
tion was completed after only 2 h from the beginning of
the experiment. In terms of statistical significance, the

TABLE 2. Contents of Selected Formulations for Physicochemical Characterization

Formulation Oil (%) Lecithin (%) Tween 80 (%) Ethanol (%) Water (q.s.)

M1 3.46 10.12 20.25 20.25 100

M2 3.45 10.81 21.64 21.64 100
M3 4.14 10.70 21.39 21.39 100
M4 2.70 9.16 18.32 18.32 100
M5 3.43 8.34 16.70 16.70 100

TABLE 3. Physichochemichal Characteristics of Selected Microemulsion Formulations

Formulation pH h (cPs) ␴ (mS/cm) DS (nm) ⫾ SD PDI ⫾ SD

M1 6.91 117.55 31.3 262.0 ⫾ 41.42 0.444 ⫾ 0.071

M2 6.98 113.35 32.7 63.10 ⫾ 6.53 0.443 ⫾ 0.036
M3 6.94 114.95 37.3 105.7 ⫾ 3.09 0.400 ⫾ 0.009
M4 6.80 81.3 36.7 21.42 ⫾ 0.2857 0.551 ⫾ 0.009
M5a 6.95 32.4 38.0 34.65 ⫾ 5.858 0.358 ⫾ 0.044
MMC-M 6.27 25.8 40.7 57.67 ⫾ 3.155 0.338 ⫾ 0.017
a
Formulation selected for drug loading.
DS, droplet size; h, viscosity; ␴, conductivity; PDI, polydispersity index; SD, standard deviation (n = 3).

Drug Dev. Res.

6 KOTMAKCHIEV ET AL.
Fig. 2. Changes in droplet size of MMC-loaded and -unloaded
microemulsions during 2-month storage at ambient temperature.
Fig. 3. Release behavior of mitomycin C from microemulsion and
mitomycin C solution. Data are given as mean ⫾ standard deviation
(n = 3).
release from MMC-M was determined to be signifi-
cantly different from the release from MMC solution
(P < 0.01) and the difference between the two groups
was also significant (P < 0.003).
Hemolytic Potential
In order to determine the hemolytic potential of
MMC-M, a modified in vitro static method was used.
Figure 4 shows the hemolytic potential of the unloaded
M, MMC-M, the MMC solution, and the surfactants at
the same ratio as that in the formulation. L showed
hemolytic activity of less than 1% at all formulation/
blood ratios that were studied. The hemolytic activity of
other surfactant T80 was found high at the employed
concentration. The MMC solution exhibited no
hemolytic activity at any of the tested doses. The
in-loaded M formulation (M5) has a low hemolytic
activity of about 0.1% at ratios 0.01, 0.05, 0.1, and 0.2.
Loading MMC into the M produced no increase in
hemolytic activity up to the formulation to blood ratio of
0.2 (Fig. 4).
Drug Dev. Res.
Fig. 4. Hemolysis induced by contents of microemulsion, MMC-
loaded and -unloaded microemulsion and MMC solution.
Cytotoxicity Study
To determine the cytotoxic effect of MMC-M and
the MMC solution on carcinoma cell lines, different
concentrations of MMC (3–300 mM) were studied. Cell
viability was determined after 24 h of incubation for
each concentration. Table 4 shows the difference
between the MMC solution and MMC-M in terms of
cytotoxicity on Calu1 and A549 cell lines, respectively.
The cytotoxic effect of MMC-M started at the first
dose and increased gradually with subsequent doses
(P < 0.05). The IC50 values of MMC-M were deter-
mined as 86.8 mM and 68.1 mM on Calu1 and A549
carcinoma cell lines, respectively. The IC50 value of the
MMC solution was determined as 181.5 mM on Calu1
cells, whereas MCM-Sol could not reach IC50 value
even at the highest concentration of 300 mM on A549
cell line (Figs. 5 and 6).
The viability of Calu1 cells that were treated with
the formulation mixtures mentioned in Table 1 were
compared with the viability of control (untreated cells)
by one-way ANOVA followed by post hoc Dunnett t
test. The viability results for mixtures C, D, F, H, and
M5 were not significantly different in comparison to
that for the control (Fig. 7). Mixtures A, B, E, and G
caused significant differences in cell viability.
DISCUSSION
M Preparation and Characterization
As shown in Figure 1, the o/w M formation area
was determined in pseudoternary phase diagram with
soybean oil, L, T80, and ethanol. Five stable systems
(Table 2) were selected by visual inspection in the o/w
M area and their physicochemical properties were
established. The viscosity of samples increased from
32 cP to 117 cP in accordance with the increase in the
amount of surfactant in the formulations (Tables 2 and
 IN VITRO CYTOTOXICITY OF MITOMYCIN C MICROEMULSION 7

TABLE 4. Comparison of Cytotoxicity of MMC-Sol and MMC-M for Each Dose Applied to Calu1 and A549 Cell Line

Calu1 % viability (SD) A549 % viability (SD)

Dose (mM) MMC-Sol MMC-M P MMC-Sol MMC-M P

3 94.372 (1.697) 91.652 (8.011) – 95.669 (3.226) 88.202 (2.752) a

15 88.357 (5.696) 79.725 (4.420) – 89.281 (2.456) 76.327 (2.654) a
30 85.330 (3.269) 65.278 (5.659) a 89.692 (0.775) 69.468 (2.902) a
90 72.814 (3.344) 56.774 (3.537) a 79.234 (3.188) 46.635 (0.352) a
150 61.922 (3.250) 45.365 (1.277) a 78.060 (2.114) 34.966 (1.313) a
210 44.233 (3.578) 34.106 (3.196) a 75.963 (3.744) 17.572 (0.831) a
300 29.024 (0.729) 16.819 (4.773) a 77.157 (1.320) 10.108 (0.708) a
a, P < 0.05; n = 3.

Fig. 5. Viability of Calu1 cell line treated with different concentra-

tions of MMC-M, MMC solution, and M after 24-h incubation. Data Fig. 7. Viability of Calu1 cells treated with different mixtures of
represent mean ⫾ standard deviation of three wells. MMC-M, micro- microemulsion components.
emulsion loaded with MMC; M5, microemulsion without MMC.

droplet size after drug loading has been reported in the

Fig. 6. Viability of A549 cell line treated with different concentra-
tions of MMC-M MMC solution and M after 24-h incubation. Data
represent mean ⫾ standard deviation of three wells. MMC-M, micro-
emulsion loaded with MMC; M5, microemulsion without MMC.
3). The conductivity of the formulations increased
slightly with the increase in water content. For further
studies, the M5 formulation was selected for drug
loading due to its lower viscosity, smaller droplet size,
lower PDI value, and lower S/Cos content. As seen in
Table 3, the droplet size of MMC-M was larger than the
droplet size of the empty M. This type of changes in
literature [Park et al., 1999; Karasulu et al., 2004; Zhang
et al., 2004; Kantarcı et al., 2007; Pestana et al., 2008].
One possible explanation for the increase in the droplet
size is that since MMC is an amphiphilic drug [Sasaki
et al., 1985], when added into the M, it interacts with
T80 and L. Formation of ion–ion or dipole–ion interac-
tions and hydrogen bonds would be expected between
the surfactants and MMC. Thus, some MMCs settle at
the interface of the M and the polyoxyethylene chains
of T80 loosen, and as a result, the droplets of MMC-M
increase in size.
As shown in Table 3, the pH values of MMC-
unloaded Ms were between 6.80 and 6.98, which is
suitable for parenteral application. Incorporation of
MMC into the M system did not significantly affect the
pH value of the M formulation. The addition of MMC
slightly increases the electrical conductivity of the
system because of its amphiphylic character.
Stability of Ms
The temperature test indicated that the devel-
oped M formulations (M1–M5) were stable under con-

Drug Dev. Res.

8 KOTMAKCHIEV ET AL.
ditions of different temperature. The storage stability
of the M with or without the drug was checked for
2 months according to droplet size measurements
(Fig. 2). After 20 days of storage, a decrease was
observed in droplet size for both M5 and MMC-M,
which may be attributed to the M undergoing slow
equilibration since it was prepared without the applica-
tion of any physical energy [Witthayapanyanon et al.,
2010]. The size of the MMC-loaded M droplet was
around 200 nm, which is much smaller than the diam-
eter (4,000–6,000 nm) that causes an increase in the
incidence of emboli [Park et al., 1999]. A further
increase in the droplet size of MMC-M after the second
20-day period may be related to further adsorption of
the drug onto the oil droplets.
In Vitro Release
As shown in Figure 3, 90% of MMC was released
from MMC solution in 1 h. MMC-M exhibited the
same amount release after 3 h. The results of statistical
analyses also showed that the release from MMC-M
was significantly different from the release of MMC
solution (P < 0.01). The slower in vitro release of MMC
from the M formulation can be explained by the parti-
tioning of MMC between the two phases in the oil–
water interface.
Hemolytic Activity
Hemolytic activity test was carried out since there
is a possibility of the passing of M droplets from the
tumor site to the systemic circulation. There are many
different methods for testing hemolytic potential of for-
mulations, employing different formulation to blood
ratios and incubation times [Krzyzaniak et al., 1997a;
Yalkowsky et al., 1998; Aparicio et al., 2005]. Figure 4
shows the hemolytic potential of the unloaded M,
MMC-M, the MMC solution, and the surfactants at the
same ratio as that in the formulation. A possible reason
for decreasing hemolytic activity of T80 in the M system
was thought to be the interaction between T80 and L at
the interface owing to the structure of the M [Jumaa
and Müller, 2000; Ishii and Nagasaka, 2004]. Earlier
studies have discussed the higher degree of hemolysis
following intravenous injection as determined by static
in vitro methods compared with the measurement by
dynamic methods due to the higher formulation : blood
ratio and longer contact time applied by the static
methods to determine hemolytic activity. Even though
the higher hemolytic activity determination represents
a disadvantage for static methods, the longer contact
time with these methods has been reported to be useful
Drug Dev. Res.
in showing the muscle damaging effects of a formula-
tion [Krzyzaniak et al., 1997b]. When a formulation
is determined nonhemolytic to slightly hemolytic, it
would mean that no change or a slight change in muscle
tissue is to be expected. Formulation : blood ratios used
in Reed and Yalkowsky’s [1985] static in vitro method
were applied in our study, which also included a ratio of
0.2, i.e., higher than 0.1, with a contact time of 30 min
instead of 2 min. Even so, hemolytic activity of
MMC-M did not exceed 1.27% in our studies. This
value is definitely lower than the 10% threshold
reported by Reed and Yalkowsky [1985] for predicting
whether a formulation is hemolytic or not (Fig. 4).
These results show that the MMC-M formulation
developed for the bronchoscopic application into the
tumor in the study is safe with regard to possible
hemolytic activity in the case of drug leakage from the
tumor to the bloodstream.
Cytotoxicity Study
As shown in Figure 5, empty M had no significant
cytotoxic effect at all doses on Calu1 cells. For A549
cells, the viability was over 81% except the 300 mM
dose, which was determined at 59% (Fig. 6). Viability
levels of Calu1 cells as determined for consecutive dose
levels of MMC in solution and those of MMC-M show
statistically significant differences (Table 5). According
to these findings, the increase in the cytotoxic effect of
MMC solution at each consecutive dose increment,
past the administration of 90 ml, showed a statistically
significant increase compared with the effect at the
preceding level. As for MMC-M application, it showed
significant differences at each level increment starting
at a dose volume of 15 ml. In A549 cells, MMC solution
showed no differences in the viability values with incre-
mental steps past that of the 90 ml dose volume, i.e.,
increasing the dose produced no additional cytotoxicity.
Application of MMC-M, on the other hand, showed
an increase in cytotoxicity with each dose increment
(Table 6).
When the effectiveness of the MMC solution
and the MMC-M were compared, it is apparent that
MMC-M exhibits higher anticancer effect than the
MMC-solution in both the Calu1 and the A549 cell
lines (Table 4). The MMC solution is nearly ineffec-
tive on the A549 cell line even at the highest dose
(77% cell viability), whereas MMC-M showed 10%
viability. As to the cytotoxicity results, MMC solution
is more potent in killing Calu1 cells rather than A549
cells (IC50 values 181.5 mM and >300 mM, respec-
tively), 35% cell viability was found with 300 mM dose
for Calu1 cells. In addition, MMC-M showed 9% cell
viability with the same dose on Calu1 cells. Based on
 IN VITRO CYTOTOXICITY OF MITOMYCIN C MICROEMULSION 9

TABLE 5. The Difference among Doses of MMC Solution (Left Side) and the Doses of MMC-M (Right Side) Applied to Calu1 Cell Line

MMC-Sol dose (mM) MMC-M dose (mM)

Dose (mM) 0 3 15 30 90 150 210 300 0 3 15 30 90 150 210 300

0
3 — —
15 a — a a
30 a — — a a a
90 a a a a a a a —
150 a a a a a a a a a a
210 a a a a a a a a a a a a
300 a a a a a a a a a a a a a a
a: P < 0.05; n = 3.

TABLE 6. The Difference among Doses of MMC Solution (Left Side) and the Doses of MMC-M (Right Side) Applied to A549 Cell Line

MMC-Sol dose (mM) MMC-M dose (mM)

Dose (mM) 0 3 15 30 90 150 210 300 0 3 15 30 90 150 210 300

0
3 — a
15 a — a a
30 a — — a a a
90 a a a a a a a a
150 a a a a — a a a a a
210 a a a a — — a a a a a a
300 a a a a — — — a a a a a a a
a: P < 0.05; n = 3.

this result, it is seen again that higher anticancer effect
is obtained with MMC-M, with IC50 values being
nearly twofold to more than fourfold higher than that
for the MMC solution on Calu1 and A549 carcinoma
cell lines, respectively.
The role of the p53 gene in tumor growth inhibi-
tion and tumor cell apoptosis is well-known [Levine
et al., 1991]. DNA damage resulting from chemo-
therapy or irradiation activates p53 and stabilizes it.
This, in turn, activates a cascade of different pathways
leading to apoptotic cell death. Tumors that encode
wild-type p53 are reported as more sensitive to chemo-
therapeutic agents than nonencoding types [Lain and
Lane, 2003]. As previously reported, MMC affected
both p53-dependent and p53-independent cell death
[Boamah et al., 2007]. The cell cultures used in this
study have different potential in developing p53 gene
expression. A549 cells express the p53 gene, while
Calu1 cells lack this property [Caamano et al., 1991].
This difference has also been confirmed by expression
analyses at the mRNA level of p53 (unpublished data).
Therefore, A549 cells should be more sensitive to
MMC action. The comparatively higher cytotoxicity, in
our study, of MMC in solution on Calu1 than on A549
cells (IC50 values 181.5 mM and >300 mM, respectively)
may be due to this difference. MMC-M, on the other
hand, had an almost equivalent cytotoxic effect on the
two cell lines, which was significantly higher than that of
the MMC solution in both cases. From this viewpoint,
MMC-M seems to be more effective than the MMC
solution in different cell types. More extensive study at
the molecular level is needed to explore the mechanism
through which the M formulation increases the effec-
tiveness of MMC on different cell cultures.
As shown in Table 1, the high toxicity of mixture G
can be explained by the absence of ethanol, the com-
ponents being unable to form an M, causing the lipo-
philic L molecules to preferably accumulate in the oil
phase leaving more T80 molecules free in the water
phase to cause high cytotoxicity (Fig. 7). For mixture D,
in which the difference from the unloaded M is the lack
of the oil phase, cell viability was determined as 94.2%.
It was thought that the reason for overcoming the cyto-
toxicity of T80 in this mixture is the formation of a
transparent micelle system. L and T80 may form mixed
micelles, in which L binds to T80 and lowers its free
concentration in the water phase. Thus, the toxic effect
of T80 is decreased. In that case, it is seen that incor-
porating soy bean oil and forming an M also masks the
toxic effect of T80, and cell viability was determined as
99.6% for the empty M. Besides, L, ethanol, and their
mixture (mixture A) and mixture F showed no toxicity

Drug Dev. Res.

10 KOTMAKCHIEV ET AL.
on Calu1 cells. Mixtures with L and without T80 (A, F,
and H) showed an increase in cell viability, which may
be attributed to a probable proliferative effect caused
by L. For the empty M (M5), the interaction between L
and T80, as explained earlier, may have occurred on the
droplet interphase and neither the toxic effect of T80
nor the proliferative effect of L was observed [Narain
et al., 1998]. The results of the cytotoxicity study con-
firmed the results of the hemolytic activity study.
Previous studies showed that intratumoral appli-
cation of antineoplastic drugs can provide highly local-
ized drug concentrations with greatly reduced systemic
toxicity [Çelikoğlu et al., 1997, 2006]. Additionally,
MMC is activated by reductive enzymes in hypoxic con-
ditions to yield its anticancer effects [Iyer and Szybalski,
1964; Bligh et al., 1990]. Therefore, the intratumoral
application of MMC may increase its in vivo efficacy in
the inner tumor tissues, where oxygenation is limited.
Besides this advantage, using M as a drug delivery
system can provide localized and slower drug release.
According to the results of the release study, nearly 70%
of MMC was released from the solution in the first half
hour, while only 30% of MMC was released from the
M. The other advantage of M is obtaining higher anti-
cancer effect with lower doses of MMC, and so, the
systemic toxicity would be lower if leakage occurred
from the tumor. Therefore, MMC-M may be suggested
as a system for providing more effective cytotoxicity on
tumor cells in intratumoral cancer therapy. In light of
the obtained results, the cytotoxic effect of MMC on
lung cancer cell lines was enhanced when it was loaded
into the M system. It is therefore proposed that the M
formulation is a potentially suitable carrier system for
the anticancer drug MMC for therapeutic purposes and
may be useful in tumor targeting for intratumoral che-
motherapy of lung cancer.
ACKNOWLEDGMENTS
This study was supported by the Scientific
Research Fund of Ege University by project number:
08.ECZ.018.
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