Synthesis of n-squalenoyl cytarabine and evaluation of its affinity with phospholipid bilayers and monolayers

Article abstract of DOI:10.1016/j.ijpharm.2010.12.038

Cytarabine (1-β-d-arabinofuranosylcytosine, Ara-C), a pyrimidine nucleoside analogue, is an attractive therapeutic agent for the treatment of both acute and chronic myeloblastic leukemias. 1,1′,2-tris-nor-Squalene acid (squaleneCOOH) has been conjugated to cytarabine with the formation of the squalenoyl-cytarabine prodrug, in order to improve the drug lipophilicity and, consequently, the affinity towards the environment of biological membranes, as well as of lipophilic carriers. The interaction of cytarabine and its prodrug with dimyristoylphosphatidylcholine (DMPC) multilamellar vesicles and monolayers has been studied by the differential scanning calorimetry and the Langmuir-Blodgett techniques. The interaction has been evaluated considering the effect of the compounds on the DMPC MLV and monolayers behaviour. The aim was to have information on the interaction of the drug and the prodrug with the biological membranes and on the possibility to use liposomes as carriers for the prodrug. The results showed an improved affinity of the prodrug with MLV and monolayers with respect to the free drug.

Full text of DOI:10.1016/j.ijpharm.2010.12.038

International Journal of Pharmaceutics 406 (2011) 69–77
Contents lists available at ScienceDirect
International Journal of Pharmaceutics
journal homepage: www.elsevier.com/locate/ijpharm
Synthesis of n-squalenoyl cytarabine and evaluation of its affinity with
phospholipid bilayers and monolayers
Maria Grazia Sarpietroa, Sara Ottimoa, Maria Chiara Giuffridaa, Flavio Roccob, Maurizio Cerutib,
Francesco Castellia,∗
a Dipartimento di Scienze Chimiche, Viale Andrea Doria 6, 95125 Catania, Italy
b Dipartimento di Scienza e Tecnologia del Farmaco, Via Pietro Giuria 9, 10125 Torino, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Cytarabine (1-␤-d-arabinofuranosylcytosine, Ara-C), a pyrimidine nucleoside analogue, is an attractive
therapeutic agent for the treatment of both acute and chronic myeloblastic leukemias. 1,1ꢀ,2-tris-nor-
Squalene acid (squaleneCOOH) has been conjugated to cytarabine with the formation of the squalenoyl-
cytarabine prodrug, in order to improve the drug lipophilicity and, consequently, the affinity towards the
environment of biological membranes, as well as of lipophilic carriers.
Received 4 October 2010
Received in revised form
20 December 2010
Accepted 27 December 2010
Available online 8 January 2011
The interaction of cytarabine and its prodrug with dimyristoylphosphatidylcholine (DMPC) multil-
amellar vesicles and monolayers has been studied by the differential scanning calorimetry and the
Langmuir–Blodgett techniques. The interaction has been evaluated considering the effect of the com-
pounds on the DMPC MLV and monolayers behaviour. The aim was to have information on the interaction
of the drug and the prodrug with the biological membranes and on the possibility to use liposomes as car-
riers for the prodrug. The results showed an improved affinity of the prodrug with MLV and monolayers
with respect to the free drug.
Keywords:
Cytarabine
n-Squalenoyl cytarabine
Prodrug
DSC
Langmuir–Blodgett
Biomembrane model
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
Han, 2007). Similar results were obtained when the long-chain fatty
acids were introduced at the 5ꢀ-hydroxyl of cytarabine (Gray et al.,
1-␤-d-Arabinofuranosylcytosine (cytarabine, Ara-C) (Fig. 1) a
pyrimidine nucleoside analogue, is a therapeutic agent to treat
both acute and chronic myeloblastic leukemias. In combination
with other anticancer agents, it is therapeutically effective against
solid tumors (Rustum and Raymakers, 1992; Grant, 1997). Its action
is presumably exerted both by inhibition of DNA polymerase and
by incorporation into DNA, which result in defective ligation or
incomplete synthesis of DNA fragments (Graham and Whitmore,
1970a,b; Major et al., 1981). Cytarabine has a low permeability
across the intestinal membrane and is rapidly deaminated to the
biologically inactive 1-␤-d-arabinofuranosyluracil in intestinal and
hepatic cells. Thus, it has a very short plasma half-life and a very
low oral bioavailability (about 20%) (Capizzi et al., 1991); as a conse-
quence the use of complex and precise dosing schedules is needed,
so its clinical utility and patient compliance are hindered.
1972). A way to provide protection against the metabolic inactiva-
tion of drugs is the encapsulation within liposomes. Nevertheless,
it has been shown that cytarabine rapidly diffuses through lipo-
somes bilayers. A way to overcome this difficulty is the chemical
modification of the drug to lipophilic prodrugs having physico-
chemical properties for incorporation into liposomes (Rubas et al.,
1986; Tokunaga et al., 1988; Schwendener and Schott, 1992).
The enhancement of the lipophilic character of drugs is a widely
applied strategy also used to overcome their difficulty in entering
cells.
We have conjugated cytarabine to the acyclic isoprenoid chain
of squalene (squaleneCOOH) obtaining the squalenoyl-cytarabine
prodrug. The squalenic moiety confers a lipophilic character to the
prodrug which should render the molecule suitable to be inserted in
phospholipid bilayers, such as a biological membrane or a liposomal
carrier. In addition, the squalenic moiety protects the NH2 group
of cytarabine from the enzymatic attach. Following previous simi-
lar approaches (Castelli et al., 2006, 2007a; Sarpietro et al., 2009),
static and kinetic experiments employing monolayers and multi-
lamellar vesicles (MLV) made of dimyristoylphosphatidylcholine
(DMPC) have been carried out to have information on the mecha-
nisms involved in the interaction of the prodrug with the biological
membranes and to have information on the use of liposomes as
carrier for the prodrug.
Much effort has been devoted to the design of prodrugs of
cytarabine, but few have led to desired products. For example,
l-valine was introduced at the NH2 group of cytarabine to syn-
thesize N4-l-valyl-Ara-C, but the oral bioavailability was only 4%
after its oral administration in rats (Cheon et al., 2006; Cheon and
∗
Corresponding author. Tel.: +39 095 221796; fax: +39 095 580138.
E-mail address: fcastelli@dipchi.unict.it (F. Castelli).
0378-5173/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijpharm.2010.12.038

70
M.G. Sarpietro et al. / International Journal of Pharmaceutics 406 (2011) 69–77
were chromatographically pure as assessed by two-dimensional
NH2
thin layer chromatography. Lipid concentration was deter-
mined by the phosphorous analysis (Rouser et al., 1970). 50 mM
tris(hydroxymethyl)-aminomethane (Tris) (pH 7.4) was used to
prepare MLV.
N
2.2. Synthesis of squalenoyl-cytarabine
HO
O
N
2.2.1. Synthesis of 1,1ꢀ,2-tris-nor-squalene acid 2:
(4E,8E,12E,16E)-4,8,13,17,21-pentamethyl-4,8,12,16,20-
O
docosapentaenoic acid
(Scheme 1)
OH
1,1ꢀ,2-tris-nor-Squalene aldehyde 1 (1.58 g, 4.12 mmol) was dis-
solved in diethyl ether (20 ml) at 0 ◦C. Separately, sulphuric acid
(2.3 ml) was added at 0 ◦C to distilled water (20 ml) with stir-
ring, followed by potassium dichromate (1.21 g, 4.12 mmol), to
obtain chromic acid. It was then added at 0 ◦C within 20 min to
the solution of aldehyde 1 previously prepared, and left to react
for 2 h at 0 ◦C with stirring. The reaction mixture was extracted
with diethyl ether (50 ml × 3), washed with saturated brine, dried
over anhydrous sodium sulfate and evaporated in vacuo. The com-
pletion of the reaction was revealed by silica gel TLC with light
petroleum/diethyl ether/methanol, 70:23:7. The crude product was
purified by flash chromatography with light petroleum, then light
petroleum/diethyl ether, 95:5 as eluant, to give 578 mg of 1,1ꢀ,2-
tris-nor-squalene acid 2 (35% yield), as a colourless oil. 1H NMR
(CDCl3): ı, 1.55–1.63 (m, 18 H, allylic CH3), 1.90–2.05 (m, 16 H,
allylic CH2), 2.26 (t, 2 H, CH2CH2COOH), 2.38 (t, 2 H, CH2CH2COOH),
5.00–5.19 (m, 5 H, vinylic CH), 12.20 (broad, 1 H, COOH). MS (EI):
m/z 400 (M+, 5), 357 (3), 331 (5), 289 (3), 208 (6), 136 (3), 81 (100).
OH
Fig. 1. Cytarabine structure.
Liposomes and monolayers have been investigated in recent
years from several points of view, ranging from their usefulness
as model biological membranes to their potential medical applica-
tions. In medicine, major interest has been focused on liposomes
as potential carriers of biological agents both in vivo and in vitro
(Pleyer et al., 1995).
The differential scanning calorimetry (DSC) has been employed
with MLV, whereas the Langmuir–Blodgett (LB) technique has been
used with monolayers. DMPC, structured as MLV, undergo a sharp
phase transition from an ordered gel-like structure (Lˇ) to a disor-
dered fluid-like structure (L˛), which is affected by the presence of
foreign molecules inside the phospholipid bilayers. DSC can detect
such a phase change and reveal the presence of the prodrug inside
the MLV (Silvius, 1991; Bach, 1994).
Langmuir–Blodgett technique is commonly used for studying
the interaction between drugs and phospholipids. Monolayers are
an excellent model for studying two-dimensional ordering, with
temperature and pressure being readily controlled (Kaganer et al.,
1999; Brezesinski and Mohwald, 2003). The analysis of the LB
results may provide important information on the organization of
biological compounds in lipid membranes (Diociaiuti et al., 2002).
The results have demonstrated that the lipophilic prodrug
exhibits a stronger affinity with the phospholipid monolayer and
bilayers than cytarabine does and that the liposomes could be used
as carrier for the prodrug.
2.2.2. Synthesis of 4-[(N)-1,1ꢀ,2-tris-nor-squalenoyl]-1-ˇ-d-
(arabinofuranosyl)cytosine (squalenoyl-cytarabine) 3:
4-[(N)-(4E,8E,12E,16E)-4,8,13,17,21-pentamethyl-4,8,12,16,20-
docosapentaenoyl]-1-ˇ-d-(arabinofuranosyl)cytosine
(squalenoyl-cytarabine) (Scheme 1)
1,1ꢀ,2-tris-nor-Squalene acid 2 (338 mg, 0.845 mmol) was dis-
solved in anhydrous THF (5 ml) in a three necked flask under
nitrogen, with stirring, followed by triethylamine (85.5 mg,
0.845 mmol) in anhydrous THF (2 ml) and it was cooled at 0 ◦C.
Ethyl chloroformate (Et-O-CO-Cl) (91.7 mg, 0.845 mmol) dissolved
in anhydrous THF (82 ml) was then added and left at 0 ◦C for
20 min, with stirring, followed by the addition of cytarabine
(Ara-C) (205.52 mg, 0.845 mmol) dissolved in anhydrous warm
DMF (5 ml), due to its low solubility. The reaction mixture was
allowed to reach room temperature and allowed to react for 3
days, with stirring, under nitrogen. The reaction mixture was
controlled by silica gel TLC with light dichloromethane/acetone,
95:5. The crude product was purified by flash chromatography
with dichloromethane, then dichloromethane/acetone, 85:15 and
finally dichloromethane/acetone, 75:25 as eluant, to give 296 mg of
2. Materials and methods
2.1. Chemicals
1H NMR spectra were recorded on a Bruker 300 ultrashield
instrument (Karlsruhe, Germany) for samples in CDCl3 solution at
room temperature, with Me4Si (TMS) as internal standard. Cou-
pling constants (J) are given in Hz. Mass spectra were recorded on
a Finnigan MAT TSQ 700 spectrometer (San Jose, CA). The reac-
tions were monitored by TLC on F254 silica gel precoated sheets
(Merck, Damstadt, Germany); after development, the sheets were
exposed to iodine vapour. Flash-column chromatography was per-
formed on 230–400 mesh silica gel. Tetrahydrofuran was dried over
sodium benzophenone ketyl. All solvents were distilled prior to
flash chromatography.
squalenoyl-cytarabine 3 (56% yield), as a colourless viscous oil. 1
H
NMR (CD3COCD3): ı, 1.55–1.68 (18 H, m, allylic CH3), 1.97–2.12
(16 H, m, allylic CH2), 2,35 (2 H, t, NHCOCH2CH2), 2.64 (2 H, t,
NHCOCH2), 3.88 (2 H, m, 5ꢀ-CH2), 4.08 (1 H, m, 4ꢀ-CH), 4.24–4.30 (2
H, m, 2ꢀ-CH and 3ꢀ-CH), 4.65–5.00 (3 H, broad peaks, OH), 5.02–5.25
(5 H, m, vinylic CH), 6.189 and 6.200 (1 H, d, J = 3.0 Hz, 1ꢀ-CH), 7.323
and 7.349 (1 H, d, J = 9.0 Hz, 5-CH), 8.187 and 8.212 (1 H, d, J = 9.0 Hz,
6-CH), 9.58–9.68 (1 H, broad peak, NHCO). 1H NMR (CD3OD): ı,
1.55–1.68 (18 H, m, allylic CH3), 1.97–2.12 (16 H, m, allylic CH2),
2.35 (2 H, t, NHCOCH2CH2), 2.54 (2 H, t, NHCOCH2), 3.90 (2 H, m, 5ꢀ-
CH2), 4.03 (1 H, m, 4ꢀ-CH), 4.10 (1 H, m, 3ꢀ-CH), 4.250 (1 H, m, 2ꢀ-CH),
5.02–5.25 (5 H, m, vinylic CH), 6.192 and 6.205 (1 H, d, J = 3.9 Hz, 1ꢀ-
CH), 7.424 and 7.449 (1 H, d, J = 7.5 Hz, 5-CH), 8.230 and 8.255 (1 H,
d, J = 7.5 Hz, 6-CH). MS (CI): m/z 627 (M+, 100), 609 (65).
Cytarabine
(1-␤-d-arabinofuranosylcytosine,
Ara-C)
(purity > 99%) was purchased from Sigma–Aldrich (Italy). Squalene
(purity > 99%) was purchased from VWR (Italy). 1,1ꢀ,2-tris-
nor-squalene aldehyde was obtained from squalene using the
method previously described (Ceruti et al., 2005 and references
therein). Synthetic l-␣-dimyristoylphosphatidylcholine (DMPC)
(purity = 99%) was obtained from Genzyme (Switzerland). Lipids

M.G. Sarpietro et al. / International Journal of Pharmaceutics 406 (2011) 69–77
71
H2SO4 , H2O
K2Cr2O7
Et2O
CHO
COOH
2
1
THF , Et3N
Et-O-CO-Cl
Ara-C , DMF
HN
C
O
N
3
O
N
HO
O
OH
H
H
H
OH
H
Scheme 1. SqualeneCOOH and squalenoyl-cytarabine synthesis.
2.3. Differential scanning calorimetry
tions (0.015, 0.03, 0.045, 0.06, 0.09, 0.12). Stock solutions of DMPC,
cytarabine and squalenoyl-cytarabine in chloroform/methanol
(1:1, v:v) were prepared. Aliquots, containing 0.010325 mmol, of
DMPC solution were distributed in glass tubes where aliquots of
cytarabine or squalenoyl-cytarabine solutions, in order to have the
chosen molar fraction, were added. The solvents were removed
under nitrogen stream at 37 ◦C. The samples were, then, freeze
dried to remove eventual solvents residues.168 ␮l of Tris solution
was added; the samples were heated at 37 ◦C for 1 min and vortexed
for 1 min, for three times and kept at 37 ◦C for 1 h.
DSC was performed by using a Mettler TA Stare equipped with a
DSC 822e calorimetric cell and a Mettler STARe V 8.10 Software. The
reference pan was filled with 120 ␮l Tris solution. The sensitivity
was automatically chosen as the maximum possible by the calori-
metric system. The calorimetric system was calibrated, in transition
temperature and enthalpy changes, by using indium, stearic acid
and cyclohexane by following the procedure of the Mettler TA
STARe Software.
2.4. MLV preparation
2.5. MLV/compounds interaction analysis
MLV were prepared both in the presence and in the absence
of cytarabine and squalenoyl-cytarabine at different molar frac-
120 ␮l of MLV was put into an aluminium calorimetric pan,
which was hermetically sealed, and submitted to the following DSC

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M.G. Sarpietro et al. / International Journal of Pharmaceutics 406 (2011) 69–77
0.12
0.09
0.06
0.045
0.03
0.015
0.00
0.12
0.09
0.06
0.045
0.03
0.015
0.00
A
B
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
°C 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
°C
Fig. 2. Calorimetric curves in heating mode, of DMPC MLV prepared in the presence of increasing molar fractions of cytarabine (A) and squalenoyl-cytarabine (B).
analysis: (i) a heating scan from 5 to 37 ◦C, at the rate of 2 ◦C/min;
(ii) a cooling scan from 37 to 5 ◦C, at the rate of 4 ◦C/min; for at least
three times. Each experiment was carried out in triplicate.
pound to eventually transfer from the loaded MLV to the unloaded
MLV; (iii) a cooling scan from 37 to 5 ◦C, at the rate of 4 ◦C/min, to
bring the phospholipid system back to the ordered state. The three
steps were repeated for at least eight times. Each experiment was
carried out in triplicate.
2.6. Monolayer measurements
3. Results and discussion
These experiments were performed by a KSV minitrough appa-
ratus provided with a computer interface unit, an operating
software, a trough (24,225 mm2 available area) made in Teflon,
two mobile barriers made in delrin. 5 mM Tris (pH 7.4) in ultra-
pure Millipore water with resistivity of 18.2 Mꢀ cm was used as
subphase. Equimolar solutions (0.001 mmol/ml) of DMPC, cytara-
bine and squalenoyl-cytarabine in organic solvents were prepared.
Mixed DMPC/compound solutions were successively prepared to
obtain for each compound the following molar fractions: 0.015,
0.03, 0.045, 0.06, 0.09, 0.12, 0.25, 0.50, and 0.75. 30 ␮l of the mixed
solutions as well as of the pure components were spread drop by
drop on the surface of the subphase by a Hamilton microsyringe;
after waiting 15 min (at the fixed temperature) to allow the solvent
evaporation, the monolayers were compressed by the use of the
two mobile barriers at the constant speed of 10 mm min−1. Sur-
face pressure vs molecular area isotherms were recorded by the
Wilhelmy plate arrangement attached to a microbalance. Before
spreading the sample, the subphase was checked twice by running
blank experiments to be sure that no impurities were present. The
experiments were performed at a subphase temperature of 10 and
37 ◦C which was kept constant by a cryothermostat connected to
the trough. Each experiment was repeated at least three times to
be sure of the results reproducibility.
One of the aims of this research was to have information on
the interaction of cytarabine and its squalenoyl-cytarabine pro-
drug with biological membranes. The second aim was to have
information on the use of liposomes as possible carrier for the
squalenoyl-cytarabine prodrug. The biological membranes are very
intricate structures and hence their biophysical interactions with
drugs are very difficult to study. For this reason, the choice of a
simplified model membrane, where all experimental parameters
can be accurately monitored, is of great importance in the study
of the elemental processes of drug–lipid interaction and several
biological membrane models have been developed. Among them,
phospholipid monolayers and liposomes were used in our research
(Maget-Dana, 1999; Peetla et al., 2009).
3.1. Synthesis of squalenoyl-cytarabine
1,1ꢀ,2-tris-nor-Squalene aldehyde 1 was obtained starting from
squalene according to a method previously developed by us (Ceruti
et al., 2005). 1,1ꢀ,2-tris-nor-Squalene acid 2 was obtained by reac-
tion of squalene aldehyde 1 with chromic acid, prepared by reacting
aqueous sulphuric acid at 0 ◦C with potassium dichromate, in 35%
yield.
Squalenoyl-cytarabine 3 was obtained according to a previously
developed method for the synthesis of squalenoyl-gemcitabine,
slightly modified (Stella et al., 2004, 2005; Couvreur et al., 2006).
Compound 3 was obtained by reacting squalene acid 2 with ethyl
chloroformate in anhydrous THF and triethylamine, followed by
addition of cytarabine, dissolved in anhydrous warm DMF, due to
its very low solubility, even in warm THF. It was allowed to react
for 3 days at room temperature; after standard work-up and chro-
matographic purification, squalenoyl-cytarabine 3 was obtained, as
a colourless viscous oil, in 35% yield.
2.7. Transmembrane transfer
60 ␮l of MLV prepared in the presence of cytarabine or
squalenoyl-cytarabine at 0.12 molar fraction (loaded MLV) was put
into the calorimetric pan and, then, 60 ␮l of MLV of pure DMPC
(unloaded MLV) was added. The pan was hermetically sealed and
submitted to the following DSC analysis: (i) a heating scan from 5
to 37 ◦C, at the rate of 2 ◦C/min, to detect the behaviour of the sam-
ples as soon as after their contact; (ii) a isotherm scan of 1 h at 37 ◦C
(above the transition temperature of DMPC), to permit the com-

M.G. Sarpietro et al. / International Journal of Pharmaceutics 406 (2011) 69–77
73
5
3.2. MLV/compounds interaction analysis
The calorimetric curve of pure DMPC MLV (Fig. 2) shows a pre-
transition peak at about 17 ◦C, due to the tilting of the hydrophobic
chains of the phospholipid, and a transition peak at about 25 ◦C,
characterized by a well defined enthalpy variation (ꢁH), due the
passage from an ordered phase (the gel phase) to a disordered phase
(the liquid-crystalline phase) (Walde, 2004). Stranger molecules in
the phospholipid bilayers of MLV can influence the phase transition
changing the temperature (Tm) at which it occurs and/or the ꢁH
(Jorgensen et al., 1991; Marsh, 1996; Castelli et al., 2007b).
MLV were prepared in the presence of different amounts of
cytarabine and squalenoyl-cytarabine and submitted to calorimet-
ric analysis. The interaction of the compounds with the MLV was
evaluated considering the variations of Tm, ꢁH, and shape of the
calorimetric curves with respect to that of DMPC MLV prepared
in the absence of compounds (Fig. 2A and B). The effect of cytara-
bine is just a small shift of the pretransition peak towards lower
temperature; whereas the transition peak remains unchanged.
Squalenoyl-cytarabine exerts a strong effect both on the pre-
transition and on the transition peaks. In fact, the pretransition
peak disappears at all the molar fractions of squalenoyl-cytarabine
present in the MLV; the transition peak broadens and shifts towards
lower temperature; moreover, starting from 0.09 molar fraction, a
phase separation occurs which indicates a not uniform distribution
of squalenoyl-cytarabine in the phospholipid bilayers and the con-
sequent formation of compound reach and compound poor regions
in the bilayers (Lambros and Rahmanb, 2004).
0
-5
-10
-15
cytarabine
A
squalenoyl-cytarabine
-20
0
0.02
0.04
0.06
molar fraction
0.08
0.1
0.12
0.14
200
0
-200
-400
-600
The transition temperature, as ꢁT/Tm0 , and the enthalpy change,
as ꢁꢁH/ꢁH0, variations of the DMPC are shown in Fig. 3A and B as
a function of the compound molar fraction in the MLV aqueous dis-
persion. The Tm, as well as the ꢁH, linearly decreases as the molar
fraction of squalenoyl-cytarabine increases. In the figure, the Tm
and ꢁH variations relative to cytarabine are shown also: almost flat
lines are obtained. The results demonstrate that cytarabine inter-
acts with the DMPC MLV very weakly, probably with the polar head
of the phospholipids producing the small shift of the pretransition
peak. This is in accord with the hydrophilic character of cytara-
bine which hindrances its insertion in the phospholipid bilayers.
On the contrary, squalenoyl-cytarabine strongly interacts with the
MLV localizing among the phospholipid molecules, probably with
the squalenoyl moiety in the acyl chain region and the cytarabine
moiety in the polar head region, and causing the fluidization of the
bilayers and the decrease of the cooperativity of the phospholipid
molecules.
cytarabine
squalenoyl-cytarabine
B
0
0.02
0.04
0.06
molar fraction
0.08
0.1
0.12
0.14
Fig. 3. Transition temperature, as ꢁT/Tm0 (panel A) and enthalpy variation, as
ꢁꢁH/ꢁH0 (panel B) of DMPC MLV prepared in the presence of increasing molar
fractions of cytarabine and squalenoyl-cytarabine as a function of the compound
molar fraction. ꢁT = Tm − Tm0 , where Tm is the transition temperature of DMPC MLV
prepared in the presence of the compound and Tm0 is the transition temperature of
pure DMPC MLV. ꢁꢁH = ꢁH − ꢁH0, where ꢁH is the enthalpy variation of MLV pre-
pared in the presence of the compound and ꢁH0 is the enthalpy variation of pure
DMPC MLV.
molar fraction, does not cause important variations on the DMPC
isotherm. For molar fraction higher than 0.12, the isotherms shift
towards lower molecular area and the LE/LC transition becomes less
3.3. Monolayer measurements
evident. Squalenoyl-cytarabine (Fig. 4B) exhibits a gas state from
2
˚
Interesting results were obtained when DMPC monolayers
were used. Single component monolayers of DMPC, cytarabine or
squalenoyl-cytarabine and mixed monolayers of DMPC and cytara-
bine and of DMPC and squalenoyl-cytarabine were spread over the
subphase. The effect of the compounds on the biomembrane model
was obtained comparing the isotherm of pure DMPC monolayer
and the isotherms of DMPC/cytarabine and DMPC/squalenoyl-
cytarabine mixed monolayers. The isotherms were recorded at
10 ◦C (below the DMPC transition temperature) and 37 ◦C (above
the DMPC transition temperature).
120 to 100 A , and a LE state at lower values of molecular area. The
addition of squalenoyl-cytarabine to DMPC causes the shift of the
isotherms towards higher values of molecular area for all the molar
fraction, with the exception of 0.75 molar fraction for which the
isotherm is shifted towards lower values of molecular area. In addi-
tion, as squalenoyl-cytarabine molar fraction increases, the LE/LC
transition becomes less evident and then it disappears.
In Fig. 5A and B the isotherms recorded at 37 ◦C are shown. At
this temperature, the DMPC isotherm is characterized by a gaseous
state between 120 and 110 A , and an LE state for lower values
of molecular area. Even at this temperature, cytarabine (Fig. 5A)
does not form any monolayer, as demonstrated by the absence of
isotherm. Its presence in the DMPC monolayer does not cause vari-
ation in the DMPC isotherm up to 0.12 molar fraction; at higher
molar fractions, the isotherms shift towards lower values of molec-
2
˚
The measures carried out at 10 ◦C are shown in Fig. 4A and B. The
isotherm of DMPC is characterized by a gaseous state from 120 to
2
2
˚
˚
100 A , a liquid expanded (LE) state from 100 to about 70 A , a liquid
2
˚
expanded/liquid condensed (LE/LC) transition from about 70 A to
2
2
˚
˚
about 44 A , and a liquid condensed (LC) state from about 44 A to
smaller values of molecular area. Cytarabine (Fig. 4A), as expected,
does not form monolayer; in fact, due to its hydrophilicity, it dis-
solves in the subphase. The addition of cytarabine, up to 0.12
ular area. Squalenoyl-cytarabine isotherm (Fig. 5B) shows a gaseous
state from 120 to 90 A , and a LE state at lower molecular area
values. The isotherms of the DMPC/squalenoyl-cytarabine mixed
2
˚

74
M.G. Sarpietro et al. / International Journal of Pharmaceutics 406 (2011) 69–77
40
30
20
10
0
100
DMPC
10 mN/m
20 mN/m
30 mN/m
A
A
0.03
0.06
0.09
0.12
0.25
0.5
0.75
80
cytarabine
60
40
20
0
0
20
40
60
80
100
120
2
molecular area (Å
)
0
0.2
0.4
0.6
0.8
1
40
30
20
10
0
DMPC
molar fraction
B
0.03
0.06
0.09
0.12
0.25
0.5
100
80
60
40
20
0
10 mN/m
20 mN/m
30 mN/m
B
0.75
squalenoyl-cytarabine
0
20
40
60
80
100
120
molecular area (Å2
)
Fig. 4. Surface pressure/molecular area isotherms of the DMPC/cytarabine (A) and
DMPC/squalenoyl-cytarabine (B) mixed monolayers, at the air–water interface at
10 ◦C.
0
0.2
0.4
0.6
0.8
1
molar fraction
40
DMPC
Fig. 6. Molecular area of DMPC/cytarabine (A) and DMPC/squalenoyl-cytarabine
(B) mixed monolayers at the air–water interface plotted as a function of the molar
fraction of compound at various values of surface pressures at 10 ◦C.
A
0.03
0.06
0.09
30
20
10
0
0.12
0.25
0.5
0.75
monolayer are shifted towards higher values of molecular area up
to 0.06 molar fraction. The isotherms with 0.09 and 0.12 molar frac-
tions of prodrug overlap the DMPC isotherm. Higher molar fractions
of squalenoyl-cytarabine cause the shift of the isotherm towards
lower values of molecular area.
cytarabine
To have information on the interaction among the molecules in
a mixed monolayer, the surface pressure/molecular area isotherms
were analyzed by calculating, at three different surface pressures
(10, 20 and 30 mN/m), the molecular area as a function of the
molar fraction of cytarabine or squalenoyl-cytarabine present in
the monolayer. The mean molecular area of a two-components
monolayer can be calculated by A = A1X1 + (1 − X1)A2, where A is
the mean molecular area, X1 is the molar fraction of component 1,
and A1 and A2 are the areas per molecule of the pure components
monolayers at the same surface pressure. Reporting in a graph A
as a function of X1, a straight line is obtained in the case the two
components are either ideally mixed or complete immiscible at
the air–liquid interface (Gaines, 1966; Shahgaldian and Coleman,
2003). Any deviation from the straight line indicates that the com-
ponents of the monolayer are miscible and exhibit a non-ideal
behaviour.
The graphs in Fig. 6A and B show the values obtained at
10 ◦C. Cytarabine causes a small positive deviation with respect
to the ideal (straight, dashed) line for all the molar fractions and
all the values of surface pressure, but less evident a 30 mN/m.
Squalenoyl cytarabine exerts strong positive deviations at all the
molar fractions and values of surface pressure. The values recorded
at 37 ◦C are shown in Fig. 7A and B. Even at this temperature,
0
20
40
60
80
100
120
molecular area (Å2
)
40
DMPC
B
0.03
0.06
0.09
0.12
0.25
0.5
30
20
10
0
0.75
squalenoyl-cytarabine
0
20
40
60
80
100
120
2
molecular area (Å
)
Fig. 5. Surface pressure/molecular area isotherms of the DMPC/cytarabine (A) and
DMPC/squalenoyl-cytarabine (B) mixed monolayers, at the air–water interface at
37 ◦C.

M.G. Sarpietro et al. / International Journal of Pharmaceutics 406 (2011) 69–77
75
100
80
60
40
20
0
squalenoyl-cytarabine interact with DMPC molecules in the mono-
layer, repulsive forces occurring among the drug or prodrug and
DMPC molecules, but with quite different intensity. In fact, these
forces are very weak in the case of cytarabine and very strong in the
case of squalenoyl-cytarabine. In the case of cytarabine, the inter-
action could be limited with the polar head of the DMPC molecules.
The decrease of the positive deviation at 30 mN/m could indicate
the high compression squeezes the cytarabine molecules out of the
DMPC monolayer, with a consequent dissolution in the subphase
and the loss of interaction with the DMPC molecules. In the case
of squalenoyl-cytarabine the interaction could involve, besides the
polar head, also the hydrophobic chains of the phospholipids at
all the surface pressures. As it is generally accepted that mono-
layers mimic bilayer behaviour at surface pressures between 30
and 50 mN/m (Jones and Chapman, 1995), the Langmuir–Blodgett
results are in agreement with those of the calorimetric experi-
ments. In fact from the calorimetric results it emerged a very weak
interaction between cytarabine and DMPC and very strong flu-
idizing effect of squalenoyl-cytarabine towards the phospholipid
bilayer.
10 mN/m
20 mN/m
30 mN/m
A
0
0.2
0.4
0.6
0.8
1
molar fraction
100
80
60
40
20
0
10 mN/m
20 mN/m
30 mN/m
B
3.4. Transmembrane transfer
Liposomes are widely recognized as an effective drug deliv-
ery system. Due to their unique characteristics, liposomes are
employed to deliver a variety of agents such as cancer therapy
drugs, antivirals, and antibiotics (Drummond et al., 1999; Allen
and Cullis, 2004; Torchilin, 2005). In addition, liposomes have been
investigated as non-viral vehicles for reaching targeted cells in gene
therapy (Noguchi et al., 1998), and as antigen carriers for immu-
nization (Thérien and Gruda, 1990). The liposome protects the drug
from metabolism and inactivation in the plasma, and, due to size
limitations in the transport of large molecules or carriers across
healthy endothelium, the drug accumulates to a reduced extent in
healthy tissues (Mayer et al., 1989).
0
0.2
0.4
0.6
0.8
1
molar fraction
Fig. 7. Molecular area of DMPC/cytarabine (A) and DMPC/squalenoyl-cytarabine
(B) mixed monolayers at the air–water interface plotted as a function of the molar
fraction of compound at various values of surface pressures at 37 ◦C.
We put in contact unloaded MLV (which mimic the biomem-
brane) and drug or prodrug loaded MLV (which mimic a loaded
lipophilic carrier) and submitted the obtained sample to DSC anal-
ysis. The calorimetric curves were recorded at intervals of one hour
and are shown in Fig. 8A and B. In figure, also the calorimetric curves
cytarabine causes small positive deviations which decrease and
almost overlap the ideal line at 30 mN/m, whereas squalenoyl-
cytarabine causes big positive deviations at all the molar fractions
and surface pressures. This indicates that both cytarabine and
0
.06
0.06
9 scan
8 scan
7 scan
6 scan
5 scan
4 scan
3 scan
2 scan
1 scan
9 scan
8 scan
7 scan
6 scan
5 scan
4 scan
3 scan
2 scan
1 scan
l
oaded
loaded
MLV
MLV
unloaded
MLV
unloaded
MLV
A
B
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
°C
°C
Fig. 8. Calorimetric curves, in heating mode, of cytarabine (A) or squalenoyl-cytarabine (B) loaded DMPC MLV left in contact with unloaded DMPC MLV at increasing time of
incubation. The curve 0.06 belongs to DMPC MLV prepared in the presence of cytarabine or squalenoyl-cytarabine at 0.06 molar fraction.

76
M.G. Sarpietro et al. / International Journal of Pharmaceutics 406 (2011) 69–77
of loaded and unloaded MLV and the curve of the MLV prepared in
the presence of compound at 0.06 molar fraction are present. The
latter is considered a reference curve. If loaded MLV lost the com-
pound, the relative peak should decrease and disappear; and if the
compound was absorbed by the unloaded MLV, the relative peak
should broaden and move towards lower temperature approaching
the reference curve. As far as cytarabine (Fig. 8A) is concerned, as
expected, all the calorimetric curves present the pretransition peak
and the transition peak which are not subjected to modification
neither in the shape nor in the position. With regard to squalenoyl-
cytarabine (Fig. 8B), in the calorimetric curve of the first scan, three
signals are present: a pretransition peak, a broad peak correspond-
ing to the loaded MLV and a well shaped peak corresponding to
the unloaded MLV. In the successive scans, the pretransition peak
decreases and disappears; whereas the broad peak and the well
shaped peak remain almost unchanged meaning that loaded MLV
do not lose the loaded compound.
Couvreur, P., Stella, B., Reddy, H., Hillaireau, H., Dubernet, C., Desmaele, D., Lepetre-
Mouelhi, S., Rocco, F., Dereuddre-Bosquet, L., Clayette, P., Rosilio, V., Marsaud,
V., Renoir, J.-M., Cattel, L., 2006. Squalenoyl nanomedicines as potential thera-
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