Interaction of 10-(octyloxy) decyl-2-(trimethylammonium) ethyl phosphate with mimetic membranes and cytotoxic effect on leukemic cells

Article abstract of DOI:10.1016/j.bbamem.2010.05.013

10-(Octyloxy) decyl-2-(trimethylammonium) ethyl phosphate (ODPC) is an alkylphospholipid that can interact with cell membranes because of its amphiphilic character. We describe here the interaction of ODPC with liposomes and its toxicity to leukemic cells

Full text of DOI:10.1016/j.bbamem.2010.05.013

Biochimica et Biophysica Acta 1798 (2010) 1714–1723
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Interaction of 10-(octyloxy) decyl-2-(trimethylammonium) ethyl phosphate with
mimetic membranes and cytotoxic effect on leukemic cells
G.A. dos Santos a,b,1, C.H. Thomé a,c,1, G.A. Ferreira a,d, J.S. Yoneda e, T.M. Nobre f, K.R.P. Daghastanli g,
P.S. Scheucher a,b, H.L. Gimenes-Teixeira a,b, M.G. Constantino e, K.T. de Oliveira h, V.M. Faça a, R.P. Falcão a,b
,
L.J. Greene a,c,d, E.M. Rego a,b, P. Ciancaglini e,
⁎
a
Instituto Nacional de Ciência e Tecnologia em Células-Tronco e Terapia Celular, São Paulo, SP, Brazil
Departamento de Clinica Médica, Faculdade de Medicina de Ribeirão Preto (FMRP-USP), 14051-140 Ribeirão Preto, SP, Brazil
Departamento de Bioquímica, Escola Paulista de Medicina, Universidade Federal de São Paulo, 04039-002 São Paulo, SP, Brazil
Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto (FMRP-USP), 14051-140 Ribeirão Preto, SP, Brazil
Departamento de Química, Faculdade de Filosofia Ciências e Letras de Ribeirão Preto (FFCLRP-USP), 14040-901 Ribeirão Preto, SP, Brazil
Instituto de Física de São Carlos (IFSC-USP), 13560-970 São Carlos, SP, Brazil
Departamento de Bioquímica, Instituto de Química (IQ-USP), 05508-900 São Paulo, SP, Brazil
Departamento de Química, Universidade Federal de São Carlos – UFSCar, Rodovia Washington Luiz, Km 235, 13565-905, São Carlos, São Paulo, Brazil
b
c
d
e
f
g
h
a r t i c l e i n f o
a b s t r a c t
Article history:
10-(Octyloxy) decyl-2-(trimethylammonium) ethyl phosphate (ODPC) is an alkylphospholipid that can interact
with cell membranes because of its amphiphilic character. We describe here the interaction of ODPC with liposomes
and its toxicity to leukemic cells with an ED-50 of 5.4, 5.6 and 2.9 μM for 72 h of treatment for inhibition of
proliferation of NB4, U937 and K562 cell lines, respectively, and lack of toxicity to normal hematopoietic progenitor
cells at concentrations up to 25 μM. The ED-50 for the non-malignant HEK-293 and primary human umbilical vein
endothelial cells (HUVEC) was 63.4 and 60.7 μM, respectively. The critical micellar concentration (CMC) of ODPC was
200 μM. Dynamic light scattering indicated that dipalmitoylphosphatidylcholine (DPPC) liposome size was affected
only above the CMC of ODPC. Differential calorimetric scanning (DCS) of liposomes indicated a critical transition
temperature (Tc) of 41.5 °C and an enthalpy (ΔH) variation of 7.3 kcal mol−1. The presence of 25 μM ODPC decreased
Tc and ΔH to 39.3 °C and 4.7 kcal mol−1, respectively. ODPC at 250 μM destabilized the liposomes (36.3 °C, 0.46 kcal
mol−1). Kinetics of 5(6)-carboxyfluorescein (CF) leakage from different liposome systems indicated that the rate and
extent of CF release depended on liposome composition and ODPC concentration and that above the CMC it was
instantaneous. Overall, the data indicate that ODPC acts on in vitro membrane systems and leukemia cell lines at
concentrations below its CMC, suggesting that it does not act as a detergent and that this effect is dependent on
membrane composition.
Received 6 January 2010
Received in revised form 21 April 2010
Accepted 12 May 2010
Available online 19 May 2010
Keywords:
10-(Octyloxy) decyl-2-(trimethylammonium)
ethyl phosphate
Lipids
Liposome
Alkylphospholipids
Leukemia
© 2010 Elsevier B.V. All rights reserved.
1. Introduction
of miltefosine is not feasible because of hemolysis and thrombophle-
bitis [4].
10-(Octyloxy) decyl-2-(trimethylammonium) ethyl phosphate
(ODPC) is a member of a new class of compounds, alkylphospholipids
(APL), which presents anticancer and antiprotozoal activity [1].
Miltefosine and edelfosine are the most prominent representatives
of this class, with miltefosine being the first drug derivative of
phospholipids clinically approved for the topical treatment of skin
metastases of breast cancer [2] and for the oral treatment of visceral
leishmaniasis [3]. However, the efficacy of oral treatment is reduced
by its gastrointestinal toxicity, which does not permit the ingestion of
a dose sufficient for systemic cancer treatment. Parenteral application
Agresta et al. [5] compared eleven new alkylphosphocholines
(APC) including ODPC to miltefosine to evaluate their hemolytic
activity and cytotoxicity on non-tumoral cells (MT2). Four com-
pounds presented lower hemolytic activity and lower cytotoxicity
than miltefosine. In subsequent experiments, of these four com-
pounds, only ODPC showed cytotoxic effects on cancer cell lines
comparable to miltefosine.
The mechanism of action proposed for this class of drugs involves
modulation of signaling pathways such as activation of SAPK/JNK and
JNK/c-JUN and inhibition of MAPK and PI3K/Akt [4,6]. In fungi, the
antiproliferative effect of edelfosine depends on selective modification
of the protein composition of cell membrane lipid rafts [7]. In human
leukemic cells, changes in the microenvironment of the cell membrane
and capping of Fas/CD95 in membrane sub-domain rafts by edelfosine
have been reported to be the mechanism that induces apoptosis [8]. Due
⁎
Corresponding author. Tel.: +55 16 36023753; fax: +55 16 36024838.
E-mail address: pietro@ffclrp.usp.br (P. Ciancaglini).
1
These authors contributed equally to this research.
0005-2736/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbamem.2010.05.013

G.A. dos Santos et al. / Biochimica et Biophysica Acta 1798 (2010) 1714–1723
1715
to their association with membranes and the modulation of diverse
signaling pathways, it is possible that APL act by inducing destabilization
of the plasma membrane, with the modulation of protein-function as a
consequence.
USA) was used for preparative chromatography. Silica gel 60 F254 plates
(Merck, Rahway, NJ, USA) were used for analytical thin layer
chromatography (TLC).
In the present study, we demonstrated the cytotoxicity of 25 μM
ODPC to three leukemia cell lines by inducing apoptosis within 24 h of
treatment and showed that this concentration was not toxic to non-
malignant cells and hematopoietic progenitor cells and determined
some physicochemical properties of ODPC. An initial characterization of
the interaction of ODPC with liposomes (large unilamellar vesicles, LUV)
was performed. Liposome-associated cancer drugs have been reported
to increase the efficacy of conventional chemotherapy by site-specific
delivery [4]. We investigated the interaction of ODPC with liposomes by
dynamic light scattering (DLS) measurements to determine the
hydrodynamic diameter of vesicular systems. Differential scanning
calorimetry (DSC) was used to determine some of the thermodynamic
parameters of these systems. Finally, carboxyfluorescein (CF) leakage
measurements were used to determinethe effect of ODPC on membrane
stability. The data reported here support the view that ODPC acts on the
membrane at concentrations far below its CMC, making the membrane
more fluid and unstable and this effect, as expected, is dependent on the
lipid composition of the membrane. Taken together, our observations
provide new information about the biological action of ODPC on models
of cell membranes and about its toxic effect on malignant cells and lack
of toxicity to normal cells at concentration up to 25 μM.
2.2. Synthesis of 10-(octyloxy) decyl-2-(trimethylammonium) ethyl
phosphate
ODPC, compound 5 (Scheme 1), was synthesized essentially as
described by Agresta et al. [5] in the original report of its synthesis
(see Supplementary material). Modifications of the synthesis were
introduced in order to optimize some of the synthetic steps. All
products obtained in each step were characterized by hydrogen
nuclear magnetic resonance (1H NMR) using a Bruker Avance
400 MHz or 500 MHz instrument. 1H chemical shifts are reported in
δ units (ppm) relative to TMS as internal standard and all spectra were
recorded in CDCl3 or CDCl3/CD3OD (2/1). All compounds were also
characterized by high resolution mass spectrometry (HRMS) ESI-TOF,
in a Q-TOF Bruker instrument.
2.3. Surface tension measurements
The critical micellar concentration (CMC) of ODPC was determined by
surface tension measurements using the pendant drop method based on a
modified axisymmetric drop shape analysis [11], with an automatic
contact-angle-tensiometer (OCA-20, Dataphysics, Bad Vilbel, Germany) as
described [12]. Drops containing different concentrations of ODPC in PBS
(137 mM NaCl, 2.7 mM KCl, 8.0 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.4)
were formed at the tip of a syringe needle in a thermostated optical glass
cuvette containing water vapor-saturated air and imaged using a CCD
camera. Software was used to trigger the recording of the images before
the formation of the complete drop. Surface tension measurements were
made on solutions of different ODPC concentrations (1.0 μM to 1.0 mM).
The surface tension was then determined by digitalizing and analyzing the
profile of the droplet and fitting it to the Young–Laplace equation. Surface
tension was considered to be at equilibrium after reaching a constant
value (b0.2 m Nm−1) for at least 2 min.
2. Material and methods
2.1. Materials
All solutions were prepared using Millipore DirectQ ultra pure
apyrogenic water. Dipalmitoylphosphatidylcholine (DPPC), phosphate-
buffered saline (PBS), Tris(hydroxymethyl)aminomethane (Tris), poli-
docanol, tetramethylsilane (TMS), CF, cholesterol (Chol), propidium
iodide (PI) and Ficoll–Hypaque were purchased from Sigma Chemical
Co. (St Louis, MO, USA). CF was converted to its sodium salt and purified
[9]. Annexin-V–FITC and BrdU flow kit were purchased from BD
Biosciences Pharmingen (San Diego, CA, USA). DNA was extracted
using the GFXTM kit purchased from GE Healthcare (Piscataway, NJ,
USA). Human recombinant cytokines (stem cell factor, FLT-3 ligand, IL-3,
IL-6, GM-CSF and erythropoietin) were obtained from Stemcell
Technologies (Vancouver, Canada), and egg-phospatidylglycerol (PG)
was purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA); egg-
phosphatidylcholine (PC) was purified as described by Maximiano et al.
[10]. All solvents and reagents used for the synthesis were purchased
from commercial sources and were analytical grade or better. Pyridine
was dried and distilled over CaH2. Ethanol-free chloroform was dried
over silica-gel. Tetrahydrofuran (THF) was dried over sodium/benzo-
phenon. N,N-dimethylformamide (DMF) was dried over CaH2, distilled
from molecular sieves (3A). Phosphorus oxychloride and triethylamine
were distilled before use. Silica gel 200–400 mesh (Acros, Trenton, NJ,
2.4. Liposome preparation
LUV composed of DPPC or DPPC:Chol (molar ratio 9:1 and 9:4) were
prepared as described below. Phospholipids were dissolved in chloroform
and dried under nitrogen. The resulting lipid film was maintained under
vacuum in a desiccator for 16 h and resuspended in PBS. The mixture was
incubated for 1 h at 60 °C, above the critical phase transition temperature
of the lipid, and vortexed for 10 min. LUV were prepared by submitting
the suspension to extrusion (eleven times) through two 100-nm
polycarbonate membranes in a LiposoFast extrusion system (Liposofast,
Sigma-Aldrich). The concentration of liposomes was 1 mg/mL in each
experiment in the presence or absence of the drug.
Scheme 1. Synthesis of 10-(octyloxy) decyl-2-(trimethylammonium)ethyl phosphate (ODPC), compound 5.

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G.A. dos Santos et al. / Biochimica et Biophysica Acta 1798 (2010) 1714–1723
2.5. Dynamic light scattering measurements
Trypan blue assay [19]. Cells were counted in a Neubauer chamber in
triplicate. ODPC was tested at 5, 10, 25 and 50 μM concentration for 24, 48
and 72 h, and PBS was used as the vehicle control. The concentration of
25 μM was selected for further experiments of apoptosis and cell
proliferation rate because of its effect on the dose screening experiments
and the lack of toxicity toward normal hematopoietic cells (see Results
section). The non-malignant adherent cells in exponential growth were
plated at a density of 105 cells per well, left to stand overnight and
subsequently treated with ODPC (10, 25, 50 and 100 μM). After 24, 48 and
72 h of treatment, the culture was photographed with an Axiovert 40
inverted microscope (Zeiss, Germany), the cells were washed with PBS,
trypsinized and counted in a T890 automatic cell counter (Beckman
Coulter, USA). The experiments were performed in triplicate.
The determinations of liposome size distribution were carried out
by DLS, using a N5 Submicron Particle Size Analyzer (Beckman Coulter,
Inc., Fullerton, CA, USA). The average value of the liposome diameters
was obtained from the unimodal distribution of DPPC liposomes
previously incubated at 25 °C, for 0 to 120 min, with 25 or 250 μM of
ODPC.
2.6. Differential scanning calorimetry
Transition phase temperatures (Tc) of the LUV membranes prepared
with different lipid compositions and in the absence or presence of ODPC
were studied by DSC. LUV suspensions and the reference buffer were
degassed under vacuum (140 mbar) for 15 min prior to use. The samples
were scanned from 20 to 100 °C at an average rate of 1 °C/min and the
recorded thermograms were analyzed using Nano-DSC II software
(Calorimetry Sciences Corporation, CSC, Lindon, UT, USA).
2.10. Apoptosis
Apoptosis was measured by three independent methods: (1)
annexin-V–PI assay, (2) morphologic evaluation and (3) DNA fragmen-
tation analysis. For the annexin-V and PI assay, 5×105 cells were
harvested by centrifugation and resuspended in 100 μL of binding buffer
(10 mM HEPES, 140 mM NaCl and 25 mM CaCl2, pH 7.4) with 5 μL of
annexin-V–FITC and 10 μL of 50 μg/mL PI, incubated for 20 min at room
temperature and analyzed by flow cytometry (FACScalibur, Becton
Dickinson Immunocytometry Systems, San Diego, CA, USA) using the
Cell Quest Software (BD BioSciences, San Diego, CA, USA). For
morphologic analysis, cytospin slides were prepared and Leishman
stained as previously described [20].
DNA fragmentation analysis was carried out as previously described
[21]. Briefly, 5×106 cells were treated with 25 μM ODPC or PBS for 3, 6, 12
and 24 h. Cells were harvested and DNA was extracted using the GFXTM kit
following the directions of the manufacturer. Nucleic acid concentration
was measured with a NanoVue nano-spectrophotometer (GE Healthcare,
USA) as absorbance at 260 nm. DNA, 5 μg/sample, was submitted to
electrophoresis in a 0.7% agarose gel.
2.7. Entrapment of carboxyfluorescein in liposomes
Liposomes containing egg-phosphatidylcholine (PC), egg-phos-
phatidylcholine:egg-phosphatidylglycerol (PC:PG 3:1 molar ratio)
or egg-phosphatidylcholine:cholesterol (PC:Chol 3:2 molar ratio)
were prepared using the extrusion system described above. CF
entrapment was carried out as described by Verly at al. [13]. Briefly,
a dichloromethane solution of each component of the lipid mixture
was added to a tube and the solvent was evaporated under a nitrogen
stream until a thin film was formed. The film was maintained under
vacuum for at least 1 h to evaporate residual solvent and then
rehydrated with 10 mM Tris–HCl buffer, pH 8.0, containing 50 mM CF.
LUV were prepared by submitting the suspension to extrusion and
free CF was removed by passing 0.25 mL of the extruded LUV through
a Sephadex-G25 medium column (1.2×20 cm) eluted with 10 mM
Tris–HCl buffer, pH 8.0 containing 300 mM NaCl. The LUV were
collected at the Vo. The phospholipid content of the eluted LUV was
determined as described by Rouser et al. [14]. Relative cholesterol
concentration is reported as % cholesterol calculated on molar basis.
2.11. Cell proliferation rate
Cell proliferation rate was measured by monitoring the incorporation
of bromodeoxyuridine (BrdU) detected by flow cytometry using the BrdU
flow kit (BD Pharmingen, USA) according to manufacturer instructions.
Cells were treated with 25 μM ODPC or PBS as vehicle control for 24 h and
exposed to 1 μM BrdU during the last 30 min of incubation, and after
processing, cells were analyzed in a FacsCalibur Flow Cytometer and
results analyzed with the Cell Quest Software.
2.8. Dye leakage measurements due to action of ODPC on LUV
membranes
Aliquots of LUV (5 μL) were added to a fluorescence cuvette
containing 0.5 mL of 10 mM Tris–HCl buffer, pH 8.0, containing
300 mM NaCl. The increase of CF fluorescence as a function of time at
25 °C at several ODPC concentrations was recorded continuously in a
Hitachi F-2000 Fluorescence Spectrophotometer (λex =490 nm and
2.12. Normal hematopoietic progenitor colony assay
λ
em =512 nm). At the end of each experiment, total CF fluorescence
was determined after the addition of 5 μL of 10% (w/v) polidocanol
which released all of the encapsulated dye. Percent CF leakage was
determined as described [13].
The study was approved by the Ethics Committees of the Faculty of
Medicine of Ribeirão Preto, USP, and of the Federal University of São Paulo,
UNIFESP (protocol no. #1129/08), and written informed consent was
obtained from all patients. Patients submitted to orthopedic surgery of the
backbone due to scoliosis were the donors. All patients (N=5) presented
normal hematologic counts and were considered to be clinically healthy
except for the orthopedic disease. Bone marrow samples were collected
by aspiration from the iliac crest. All samples presented a normal
distribution of hematopoietic cells as evaluated by morphologic analysis
(myelogram). Mononuclear cells were isolated by density gradient Ficoll–
Hypaque centrifugation and counted in a Newbauer chamber. Cells were
diluted in methylcellulose medium supplemented with human recombi-
nant cytokines (stem cell factor, FLT-3 ligand, IL-3, IL-6, GM-CSF and
erythropoietin) and plated onto 35-mm gridded plates. ODPC (25 or
50 μM final concentration) or PBS vehicle was added to the medium.
Burst-forming units—erythroid (BFU-E), colony-forming units—erythroid
(CFU-E) and colony-forming units—granulocyte and macrophage
(CFU-GM) were counted after 14 days of incubation at 37o C, 5% CO2.
2.9. Cell culture, cell counting and viability measurements
The human cell lines NB4 (acute promyelocytic leukemia) [15], U937
(histiocytic lymphoma with myeloid markers) [16] and K562 (chronic
myeloid leukemia in blast crisis) [17] were cultured at 37 °C with 5% CO2
in RPMI 1640 medium supplemented with 10% fetal bovine serum. The
non-malignant embryonic cell line HEK-293 was cultured in DMEM
medium supplemented with 20% fetal bovine serum. Primary human
umbilical vein endothelial cells (HUVEC) were obtained as previously
described by Covas et al. [18] with modifications, i.e., cells were cultured in
EGM-2 medium (Lonza, USA) supplemented with 20% fetal bovine serum
to avoid cell differentiation. All other cell lines were acquired from ATCC.
Cell viability measurements of the leukemia lineages were performed
with an initial minimum viability of at least 95% as determined by the

G.A. dos Santos et al. / Biochimica et Biophysica Acta 1798 (2010) 1714–1723
1717
3. Results
toxic to non-malignant cells or to normal hematopoietic progenitors
(Fig. 8). The second 250 μM, is above the CMC of ODPC.
3.1. Synthesis of 10-(octyloxy) decyl-2-(trimethylammonium) ethyl
phosphate
Dynamic light scattering (DLS) showed that DPPC-liposome size was
not affected by the presence of 25 μM ODPC for up to 2 h of incubation
(Fig. 2), and there were no changes in PI. However, in the presence of
250 μM ODPC, there was an increase of liposome size from 150 to
600 nm after 1 h of incubation. It is important to note that the number of
liposomes with increased diameter decreased after 1 h, probably due to
their rupture. The light scattering data also indicated an increase of PI for
the liposome system, with a reduction in the scattering intensity, after
1 h in thepresence of 250 μM ODPC. PI=0.98 indicated heterogeneity of
the liposome size range. When the same study was carried out with a
liposome system prepared with different proportions of cholesterol
(DPPC:cholesterol 9:1 or 9:4 molar ratio), the liposome diameters were
not affected by either 25 or 250 μM ODPC.
In order to determine if the insertion of ODPC into the membrane
had really occurred, a DSC assay was performed. Fig. 3 shows the DSC
thermograms of liposomes of different compositions in the absence
and presence of 25 and 250 μM ODPC, concentrations below and
above the CMC. Only one transition temperature was observed for all
systems. When ODPC was present at the concentration of 250 μM,
there was a slight displacement of the Tc to lower values: 4.6, 1.3 and
0.1 °C for DPPC; DPPC:Chol (9:1) and DPPC:Chol (9:4), respectively.
The data in Table 1 indicate that the variation of enthalpy (ΔHcal) of
ODPC and liposomes of different compositions decreased with
increasing ODPC concentration, irrespective of liposome composition,
showing that ODPC induced thermodynamic modifications in the lipid
bilayers. This phenomenon is also demonstrated in Fig. 3, where
increased concentrations of ODPC reduced the heat capacity of the
liposomes in a concentration-dependent manner.
ODPC was obtained as indicated in Scheme 1. Compound 2 was
synthesized by monoprotectionof decanodiol(compound1)generating
the mono-alkoxide in DMF/NaH followed by the reaction with benzyl
chloride and purified on a silica-gel column (65% yield relative to the
previous synthetic step). Then, compound 2 was alkylated with
1-bromo-octane after alkohoxide generation (NaH/THF), giving com-
pound 3 at 79% yield after purification on a silica-gel column. Com-
pound 3 was deprotected with hydrogen and a C/Pd catalyst, providing
compound 4 at 76% yield after purification on silica-gel column. The last
step was the addition of choline performed by reacting compound 4
with POCl3 and Et3N/CHCl3 and then with choline tosylate in warm
pyridine (50 °C). Water was then added. Compound 5 was purified
using CH3OH:acetic acid 6:4 as eluent (55% yield) on a silica-gel column.
The overall yield was 21% for compound 5. Mass spectrometry indicated
a signal at 474.3319 U, which corresponds to [Molecule+Na] ion, and
the signal at 490.3056 U, which corresponds to [Molecule+K] ion. Both
adducts were formed during the analysis and do not indicate
contamination of the ODPC. 1H-NMR analysis confirmed the homoge-
neity and identity of compound 5. Details of the synthesis are available
in Supplementary material.
3.2. Determination of the critical micellar concentration of ODPC
The plot of surface tension versus ODPC concentration is given in
Fig. 1. Increasing ODPC concentration decreased the surface tension.
However, at approximately 200 μM ODPC, the surface tension became
constant, indicating micelle formation in the bulk. Extrapolating the
two lines indicates that the critical micellar concentration of ODPC
was 200 μM.
The composition of the liposomes had an even greater effect on
ΔHcal when one considers only the concentration of 25 μM (far below
the CMC of 200 μM). In liposomes composed of DPPC:cholesterol
(9:4), a composition closer to that present in biological systems, the
effect on ΔHcal was more pronounced. In addition to DPPC (Fig. 3A)
and DPPC:Chol (9:1) (Fig. 3B), the width at half height of peak (ΔT1/2
)
3.3. Interaction of ODPC with liposomes
was greater when ODPC was present at a concentration above the
CMC (200 μM) (Table 1). However, this increase was not observed
with DPPC:Chol (9:4) (Fig. 3C), a relative concentration of cholesterol
that led to a significant broadening of the peaks, thus forming systems
less stable than the others.
We studied ODPC at two concentrations to better determine the
mechanism of interaction of ODPC with membranes and its possible
dependence on a detergent action. The first, 25 μM, was chosen because
it was effective on leukemic cells (see details below in Figs. 5–7) and not
Fig. 1. Effect of ODPC concentration on surface tension. The surface tension measurements
were carried out in 5 mM Tris–HCl buffer, pH 7.0, containing 150 mM KCl at 25 °C. Data are
reported as means SD for 5 measurements per point. The critical micellar concentration
was 200 μM ODPC.
Fig. 2. Effect of ODPC on liposome diameters. Data are reported as means SD for 3
measurements. Average values of the liposome diameters were obtained by dynamic light
scattering (unimodal distribution) of DPPC liposomes incubated at 25 °C for different
periods of time with (○) 25 and (●) 250 μM ODPC.

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G.A. dos Santos et al. / Biochimica et Biophysica Acta 1798 (2010) 1714–1723
Fig. 4. Effect of ODPC on carboxyfluorescein leakage from liposomes of different
compositions. The concentration of added ODPC (arrow) was 100 μM and the liposome
composition was PC (___); PC:PG 3:1 molar ratio (- - -); PC:Chol 3:2 molar ratio (……).
Arrows indicate the time of addition of ODPC and polidocanol, respectively.
CF from PC LUV within 18 min. Other ODPC concentrations were
tested (200, 400 and 600 μM) and showed similar leakage rates for the
same period independently of the ODPC concentration (Table 2). It is
important to note that even when using an ODPC concentration at or
above the CMC (3-fold CMC), the leakage was not complete during the
20 min studied.
The effect of the negative charge of the liposome membrane on the
permeabilization activity of ODPC was studied using LUV consisting of
PC:PG (3:1 mol ratio) and monitoring the increase of CF fluorescence
during its leakage (Fig. 4). For 100 μM ODPC, the CF leakage was 39%
within 18 min. The permeabilization caused by the addition of 400 μM
ODPC was about 48% as summarized in Table 2. These results suggest a
decrease of membrane permeability in the presence of a negative
charge. Also, an expressive decrease (17%) in CF leakage to 100 μM
ODPC was observed for PC:Chol (3:2) (a concentration below CMC)
compared to the other LUV preparations (Table 2).
Fig. 3. Differential scanning calorimetry (DSC) thermograms of liposomes in the absence and
in the presence of ODPC. DSC thermograms were determined using an N-DSC II calorimeter
and the result is reported as Cp (kcal/K.mol) as a function of temperature (°C). The liposomes
were (A) DPPC, (B) DPPC:Cholesterol 9:1 molar ratio and (C) DPPC:Cholesterol 9:4 molar
ratio, in the absence (____) and in the presence of 25 (- - -) and 250 μM (……) ODPC.
3.4. Membrane permeabilization
3.5. Cytotoxic screening assays (cell counting and viability) on leukemic
cells
The rate of CF leakage from the vesicles was measured by the
increase of fluorescence emission as a function of time in order to
study the membrane permeabilization after ODPC insertion. CF is self-
quenched at high concentrations such as that trapped inside the
liposomes used in these experiments. Fig. 4 shows the kinetics of CF
leakage from PC, PC:PG (3:1) and PC:Chol (3:2) LUV. CF leakage of less
than 4% ODPC was observed for all liposome systems studied when up
to 50 μM ODPC was used or when ODPC was absent (see Table 2).
All kinetic curves were obtained after the addition of 100 μM ODPC
(below the CMC). This ODPC concentration promoted a 59% leakage of
In order to determine the concentration at which ODPC was
effective, we measured cell proliferation and viability using three
representative cell lines of acute myeloid leukemia, i.e., NB4, U937 and
K562. In a screening experiment, treatment with 25 μM ODPC for 24 h
induced a decrease of approximately 50–60% in the proliferation of
NB4 and U937 cell lines (Fig. 5). The K562 cell line was more resistant
in the first 24 h (Fig. 5). However, after 48 and 72 h of treatment, the
three cell lines showed similar sensitivity and a low viability rate in
response to 25 μM ODPC even at lower ODPC concentrations (Fig. 5).
Table 3 presents the estimated ED-50 for each cell line at 24, 48 and
72 h.
Table 1
Effect of ODPC concentration and lipid composition on thermodynamic values
describing the interaction with liposomes.
Table 2
CF Leakage from LUV composed of PC, PC:PG (3:1 molar ratio) and PC:Chol (3:2 molar
ratio) in the absence or presence of ODPC.
Liposomes
ODPC Tc
(μM) (°C)
ΔHcal (kcal mol−1 ΔH decrease (%)a ΔT1/2 (°C)
)
ODPC
(μM)
CF leakage (%)
PC
DPPC
0
25
250
0
25
250
0
41.5 6.80
41.5 4.44
36.9 0.483
41.0 11.5
40.9 3.03
39.7 0.329
45.0 9.95
45.1 1.50
44.9 1.01
—
1.60
1.70
1.95
1.65
1.64
2.69
13.8
13.8
13.4
34.7
92.9
—
PC:PG
PC:Chol
0
25
50
100
200
400
600
0.03
2.06
3.52
58.8
62.9
61.5
64.1
0.02
1.11
1.12
39.2
50.1
48.9
44.6
0.02
1.92
3.22
17.0
23.3
18.9
17.7
D P P C : C h o l
(9:1)
73.7
97.1
—
D P P C : C h o l
(9:4)
25
250
85.0
89.8
a
Decrease of ΔH with respect to the control (absence of ODPC).

G.A. dos Santos et al. / Biochimica et Biophysica Acta 1798 (2010) 1714–1723
1719
Fig. 5. ODPC reduces the proliferation and viability of cultured leukemia cells. Cells in exponential growth were plated at a concentration of 5×105 cells/ml and treated with ODPC or PBS
vehicle. In the upper panels, NB4 (acute promyelocytic leukemia); central panels, U937 (histiocytic lymphoma with myeloid markers); lower panels, K562 (chronic myeloid leukemia in
blast crisis). The concentrations of ODPC are indicated by the symbols and time is given on the abscissa. In the left row, cell proliferation was determined by cell counting in a Neubauer
chamber. The results are reported as percent inhibition of proliferation compared to control. In the right row, viability was evaluated by Trypan blue exclusion dye assay. The experiments
were carried out in triplicate and data are reported as mean SD. When error bars are not given, they are smaller than the symbols.
Table 3
Effective dose (ED) 50% of ODPC for the inhibition of the proliferation of leukemic and non-malignant cells.
24 h
48 h
72 h
ED-50 (μM)
95% CI
R2
ED-50 (μM)
95% CI
R2
ED-50 (μM)
95% CI
R2
NB4
U937
K562
HEK-293
HUVEC
22.12
13.89
186.4
99.49
98.06
19.35 to 25.30
11.81 to 16.34
132.3 to 262.4
90.56 to 109.3
87.77 to 109.6
0.9617
0.9466
0.9596
0.9575
0.9474
6.737
7.581
10.43
75.58
73.75
5.930 to 7.653
7.372 to 7.796
8.813 to 12.34
72.09 to 79.24
68.47 to 79.43
0.9663
0.9977
0.9419
0.9898
0.9762
5.357
5.629
2.937
63.44
60.68
4.857 to 5.909
5.509 to 5.751
2.548 to 3.386
58.65 to 68.61
55.11 to 66.82
0.9726
0.9976
0.9809
0.9787
0.9689
95% CI is the 95% confidence interval for ED-50, expressed as μM.
R2 is the goodness of fit coefficient.

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G.A. dos Santos et al. / Biochimica et Biophysica Acta 1798 (2010) 1714–1723
3.6. Anti-proliferative action of ODPC and induction of apoptosis in leukemic
cells
BrdU (Fig. 6), a marker of DNA synthesis and S phase of the cell cycle,
confirmed a high sensitivity of the leukemic cell lines to ODPC as
demonstrated in Fig. 4. The assays of apoptosis (phosphatidylserine
externalization and DNA fragmentation analysis) revealed the same
tendency. An extensive DNA fragmentation pattern was detected as
After 24 h, 25 μM ODPC induced apoptosis and inhibited the
proliferation rate of NB4 and U937 cell lines. The incorporation of
Fig. 6. Proliferation rate and apoptosis of culturedleukemia cells treated with ODPC. Cells in exponential growth were plated ata concentration of 5×105 cells/ml and treatedwith ODPC or
PBS vehicle for 24 h or the indicated periods of time. Upper panel, NB4; middle panel, U937; lower panel, K562. The cells weretreated with 25 μM ODPC for the times indicated in the figure.
Proliferation rate was evaluated by BrdU incorporation (left row), and cell cycle analysis was carried out with the DNA-specific dye 7-AAD. The gates show the cell cycle phases G1, S and
G2/M. In the middle panel, apoptosis was evaluated by phosphatidylserine externalization, with cells positive for annexin-V considered to be apoptotic. Numbers indicate the percent of
cells in each gate. DNA fragmentation electrophoretic analysis is shown in the right row. Data are representative of three independent experiments.

G.A. dos Santos et al. / Biochimica et Biophysica Acta 1798 (2010) 1714–1723
1721
early as 6 h after treatment with 25 μM ODPC in the more sensitive
cell lines NB4 and U937 compared to K562 (Fig. 6). We confirmed the
pro-apoptotic action of ODPC also by morphologic analysis that
showed pycnotic nuclei and membrane blebbing in a significant
fraction of cells (Fig. 7).
reduction of approximately 50% in the number of colonies in relation
to control (Fig. 8C).
4. Discussion
It has been proposed that the APL family of drugs interacts with
cell membranes due to their amphiphilic character; however, the
exact mechanism and the possible dependence on a detergent action
have not been clarified. Their probable alignment at interfaces reflects
the tendency of ODPC to assume the most energetically favored
orientation and to act as a detergent. Detergent-based cell lysis (and
consequent death) is a general phenomenon that would not be
expected to be specific and toxic only to cancer cells. Indeed, this non-
specific mechanism may be responsible for the gastrointestinal
toxicity of miltefosine at high doses [3].
3.7. Effect of ODPC on non-malignant cells
ODPC at concentrations up to 25 μM was not toxic to the growth of
HEK-293 (embryonic kidney cells, Fig. 8A) and to a primary HUVEC
(Fig. 8B); however, the concentrations of 50 and 100 μM showed
toxicity to both cells. Microscopic evaluation confirmed the results
(Supplemental Figs. 1 and 2). Estimated ED-50 for both cells at 24, 48
and 72 h treatment are presented in Table 3.
3.8. Effect of ODPC on normal hematopoietic progenitors
In order to determine if the toxicity of ODPC against cancer cells
was dependent on a detergent action, we first evaluated its CMC. ODPC
presented a CMC of 200 μM determined by surface tension measure-
ments. The compound closest to ODPC in structure found in the
literature is octadecyl trimethyl ammonium chloride (OTAC), which
also contains 18 carbons in the alkyl chain. This cationic amphiphilic
compound has a CMC of 190 μM [12], which is close to that of ODPC,
200 μM. Comparing OTAC and ODPC, their equivalent CMCs indicate
that the polar head groups can present similar size and the extension of
the alkyl chain is determinant for micelle formation.
We performed a methylcellulose-based hematopoietic progenitor
colony assay to evaluate the toxicity of ODPC to normal hematopoietic
cells. ODPC (25 μM) did not change the proliferation or differentiation
of normal hematopoietic progenitors as shown in Fig. 8C. However,
the concentration of 50 μM was toxic to these cells and induced a
The results of biological assays demonstrated that 25 μM ODPC was
toxic to leukemic cells but not to normal hematopoietic cells. At this
point it is important to notice that methylcellulose-based hematopoi-
etic colony assay is a validated procedure that is capable of predicting
the maximum tolerated dose in vivo [22–24]. The demonstration of
apoptosis excludes a cytostatic effect in the treatments with 25 μM
ODPC. Apoptosis is a common consequence of exposure of cancer cells
to effective chemotherapeutic agents [25,26]. In addition, the results
with the embryonic kidney cell line HEK-293 and with HUVEC
demonstrate that ODPC, at concentrations that are toxic to leukemia
cells, spares non-malignant cells of a non-hematopoietic origin.
Our results can be compared with those obtained by Agresta et al. [5]
who estimated the ED50 for 48 h treatment of ODPC at more than 100 and
68 μM for inhibition of proliferation of K562 cells using the MTT and 3H
thymidine assays, respectively. In the present study, we have estimated
the same ED50 at 10.43 μM but we have used another method, i.e.,
microscopic cell counting. Differences in the methods used or in the basal
state of growth of the cell line may explain this discrepancy.
To characterize ODPC, we evaluated its interaction with liposomes
that are used as models for the study of biological membranes.
Liposomes have many physicochemical properties similar to those of
cell membranes such as membrane permeability, osmotic activity and
interaction with various solutes, surface characteristics and chemical
composition [27]. The fluidity of their membranes and their self-
closing structure are essential parameters for the study of biological
membrane function. The DSC measurements of thermodynamic
parameters of the interaction with liposomes are important to identify
changes of structural and physicochemical characteristics of lipid
bilayers caused by drugs [27]. With this model system, we were able to
obtain several indications of how ODPC disturbs liposome structure.
Our results indicated that the incorporation of ODPC into DPPC and
DPPC:Chol bilayers (Fig. 3) caused a reduction in the variation of ΔHcal
and Tc, indicating that ODPC destabilized the liposome (Table 1). The
decrease of ΔHcal was greater when ODPC was incorporated into the
lipid bilayer at a concentration above the CMC, indicating that this
modification was concentration dependent. Moreover, 25 μM ODPC, a
concentration demonstrated to be cytotoxic to leukemic cells and far
below the CMC, induced a significant reduction of ΔHcal in relation to
PBS vehicle control. Furthermore, this effect was dependent on the
lipid composition of liposomes and was greater when cholesterol was
present in the preparation (Table 1). This result is consistent with the
Fig. 7. Morphologic analysis of the effect of 25 μM ODPC on cultured leukemia cells. Cells in
exponential growth were plated at a concentration of 5×105 cells/ml and treated with ODPC
or PBS vehicle for 24 h. In the upper panels, NB4 (acute promyelocytic leukemia); central
panels, U937 (histiocytic lymphoma with myeloid markers); lower panels, K562 (chronic
myeloid leukemia in blast crisis). The vehicle control is in the left row and cells treated with
25 μM ODPC are in the right row. The characteristic membrane blebbing and the nuclear
condensation, morphologic hall markers of apoptosis, are seen especially in the NB4 and
K562 samples treated with ODPC. Representative experiment of three independent
experiments.

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G.A. dos Santos et al. / Biochimica et Biophysica Acta 1798 (2010) 1714–1723
Fig. 8. Evaluation of ODPC toxicity to non-malignant cells and to normal hematopoiesis. (A) Embryonic kidney cells (HEK-293) and (B) primary human umbilical vascular endothelial
cells (HUVEC) were cultured and treated as described in Section 2.9. The concentrations of ODPC are indicated by the symbols and time is given on the abscissa. Results are reported
as a normalized inhibition of proliferation in relation to the PBS-treated control. (C) The procedure of methylcellulose-based hematopoietic colony assay from normal donor bone
marrow samples is described in Section 2.12. Colonies were counted after 14 days. Number of colonies was normalized to control (PBS vehicle) as an ODPC/control ratio, with values
significantly lower than 1 indicating toxicity. BFU-E: burst-forming units—erythroid; CFU-E: colony-forming units—erythroid; CFU-GM: colony-forming units—granulocyte and
macrophage. *Pb0.05 compared to control (one-way ANOVA followed by Dunnett's multiple comparison test between control and 25 or 50 μM treated samples for each type of cell).
hypothesis that APL may preferentially interact with highly organized
sub-domains named rafts [28] that are rich in cholesterol [29]. Indeed
it has been proposed that normal raft assembly may be essential to
signaling transduction in the plasma membrane [30].
progressive increase in CF leakage (Fig. 4). Using ODPC concentrations
above 200 μM, an instantaneous leakage occurred, which was constant
during the experiment (data not shown). This effect was probably due to
the fact that the ODPC molecule inserted into the membrane caused a
transitory perturbation and fast leakage. The membrane then returns to
equilibrium and the leakage becomes slower or stops. Furthermore, in
negative membranes, the total CF leakage during the same period was
smaller than that observed for PC LUV. This effect could be due to (i)
columbic interactions among the polar phospholipid head groups that
reduced membrane leakage or (ii) the high hydration in the interface
region caused by the negative charges present in the phosphate group.
In addition to the negative charge effect, the permeabilization action of
ODPC was also studied in the presence of a membrane prepared with a PC:
Chol 3:2 molar ratio (Table 2), a condition proposed to be the closest to the
erythrocyte membrane, with 41 mol% cholesterol [13,32]. In this case, any
increase of ODPC concentration (Table 2 and Fig. 4) added to membrane
samples had less effect on membrane permeabilization compared to PC
and even PC:PG LUV. Actually, it is well known that the presence of
cholesterol can change the lipid packing and increase its rigidity. This
result is consistent with the report of Agresta et al. [5] who described
The strong effect on the enthalpy in the transition indicates that
the ODPC must be inside the hydrophobic interior of the phospholipid
array and not superficially in the vicinity of the polar groups or at the
interface of the phospholipids [31]. Furthermore, Table 1 shows that
the ΔT1/2 for 250 μM ODPC is greater than that for 25 μM ODPC and
that this increase is more significant for DPPC:Chol at a molar ratio of
9:1, indicating that the presence of cholesterol leads to greater
destabilization, decreasing the cooperativity of the system. Fig. 3C
shows that cholesterol at a molar ratio of 9:4 led to a significant
broadening of the peaks and the incorporation of ODPC did not change
ΔT1/2 values. Therefore, cholesterol was responsible for the destabi-
lization of phospholipid assemblies.
To further study the properties of ODPC related to the hemolytic
activity of APC, we performed studies of permeabilization in membrane
systems composed of PC, PC:PG or PC:Chol. Only at ODPC concentrations
below the CMC did the leakage present a kinetic behavior with a

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ODPC as an APL with minor hemolytic activity, a result that may represent
a significant advantage in future clinical trials.
In the present study, we demonstrated that ODPC induced cell
death primarily by apoptosis and inhibited proliferation in three
human leukemia cell lines in the concentration range of 10 to 50 μM
that is far below the CMC. Interestingly, Agresta et al. [5] determined
the IC-50 of ODPC against MT2 cells (a non-tumoral cell line) to be
greater than 100 μM, and the hemolytic activity for the same
compound was demonstrated only with concentrations above 2 mM.
The physicochemical data obtained in the present study indicate that
the interaction of ODPC with mimetic PC membranes occurs even below
the CMC. DLS, DSC and CF leakage revealed that the insertion of ODPC into
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Acknowledgements
The authors thank Prof. Dr. Stefano Servi, Dipartimento di Chimica,
Materiali, Ingegneria Chimica “Giulio Natta,” Politecnico di Milano and
CNR, Istituto di Chimica del Riconoscimento Molecolare “Adolfo Quilico,”
Italy, for providing a sample of the original compound that was used as a
standard for comparison with ODPC synthesized in our laboratory and
Maristela D. Orellana and Alexandre Krause for technical assistance with
HUVEC cells and HEK-293 cells, respectively. The authors also thank the
following Brazilian governmental agencies for financial support: Conselho
Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coorde-
nação de Aperfeiçoamento do Pessoal de Ensino Superior (CAPES),
Fundação de Amparo a Pesquisa do Estado de São Paulo (2008/06619-4)
and Fundação de Apoio ao Ensino, Pesquisa e Assistência do Hospital das
Clínicas da FMRP-USP (FAEPA). Thanks are also due to Prof. Norberto
Pepoline Lopes for High Resolution Mass Spectroscopy (ESI) analyses.
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Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at doi:10.1016/j.bbamem.2010.05.013.
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